Method to provide bacterial ghosts provided with antigens

ABSTRACT

Methods for improving binding of a proteinaceous substance to cell-wall material of a Gram-positive bacterium are disclosed. The proteinaceous substance includes an AcmA cell-wall binding domain, homolog or functional derivative thereof. The method includes treating the cell-wall material with a solution capable of removing a cell-wall component such as a protein, lipoteichoic acid or carbohydrate from the cell-wall material and contacting the proteinaceous substance with the cell-wall material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application NumberPCT/NL02/00383 filed on Jun. 11, 2002, designating the United States ofAmerica, and published in English, as PCT International Publication No.WO 02/101026 A2 on Dec. 19, 2002, the contents of the entirety of whichare incorporated by reference.

TECHNICAL FIELD

The present invention pertains to a method for obtaining cell-wallmaterial of Gram-positive bacteria with an improved capacity for bindinga proteinaceous substance comprising an AcmA cell-wall binding domain,as well as pharmaceutical compositions including the obtained cell-wallmaterial.

BACKGROUND

Heterologous surface display of proteins (Stahl and Uhlen, TIBTECH May1997, 15, 185–192) on recombinant microorganisms via the targeting andanchoring of heterologous proteins to the outer surface or the cell wallof host cells, such as yeast, fungi, mammalian cells, plant cells, andbacteria, has been possible for several years. Display of heterologousproteins at the surface of these cells has taken many forms includingthe expression of reactive groups such as antigenic determinants,heterologous enzymes, single-chain antibodies, polyhistidyl tags,peptides, and other compounds. Heterologous surface display has beenapplied as a tool for applied and fundamental research in microbiology,molecular biology, vaccinology and biotechnology. Another application ofbacterial surface display has been the development oflive-bacterial-vaccine delivery systems. The cell-surface display ofheterologous antigenic determinants has been considered advantageous forinducing antigen-specific immune responses in live recombinant cellsused for immunization. Another application has been -the use ofbacterial surface display in generating whole-cell bioadsorbents orbiofilters for environmental purposes, microbiocatalysts, and diagnostictools.

Generally, chimeric proteins include an anchoring or targeting portionthat is specific and selective for the recombinant organism, wherein theanchoring portion is combined with the reactive group, such as theantigenic determinant, heterologous enzyme, single-chain antibody,polyhistidyl tag, peptide, or other compound. A well known anchoringportion comprises the so-called LPXTG (SEQ ID NO: 1) box, whichcovalently binds to a Staphylococcus bacterial surface, i.e., in theform of a fully integrated membrane protein. In this manner, at leasttwo polypeptides of different genetic origins may be joined by a normalpeptide bond to produce a chimeric protein. For example, PCTInternational Patent Publication No. WO 94/18330, which relates to theisolation of compounds from complex mixtures and the preparation ofimmobilized ligands (bioadsorbents), discloses a method for obtaining aligand comprising anchoring a binding protein in or at the exterior of acell wall of a recombinant cell. The binding protein is essentially achimeric protein produced by the recombinant cell and includes anN-terminal part derived from an antibody that is capable of binding to aspecific compound, wherein the N-terminal part is joined to a C-terminalanchoring part, derived from an anchoring protein purposely selected forbeing functional in the specific recombinant cell chosen. PCTInternational Patent Publication No. WO 97/08553 discloses a method forselectively targeting proteins to the cell wall of Staphylococcus sp.,using anchoring proteins which include long stretches of at least 80–90amino acid long amino acid cell-wall-targeting signals. The signals arederived from the lysostaphin gene or amidase gene of Staphylococcus andencode for proteins that selectively bind to Staphylococcus cell-wallcomponents.

Vaccine delivery or immunization systems with attenuated bacterialvector strains that express distinct antigenic determinants against awide variety of diseases are currently being developed. Mucosal vaccinesfor nasal or oral passages using these attenuated bacterial vectors havereceived a great deal of attention. For example, both systemic andmucosal antibody responses against an antigenic determinant of hornetvenom have been detected in mice orally colonized with a geneticallyengineered human oral commensal Streptococcus gordonii strain thatexpresses the hornet venom antigenic determinant on its surface(Medaglini et al., PNAS 1995, 2; 6868–6872). A protective immuneresponse was also elicited by oral delivery of a recombinant bacterialvaccine that included tetanus toxin fragment C constitutively expressedin Lactococcus lactis (Robinson et al., Nature Biotechnology 1997, 15;653–657). Mucosal immunization is considered an effective means ofinducing IgG and secretory IgA antibodies directed against specificpathogens of mucosal surfaces.

Immunogens expressed by bacterial vectors may be presented in aparticulate form to antigen-presenting cells, such as M-cells, of theimmune system and therefore should be less likely to induce tolerancewhen compared to soluble antigens. Additionally, the existence of acommon mucosal immune system permits immunization of one specificmucosal surface in order to induce secretion of antigen-specific IgA andother specific immune responses at distant mucosal sites. A drawback tousing bacterial vectors for immunization is the potential of thebacterial strain causing inflammation or disease and potentially leadingto fever or bacteremia. Instead of using attenuated bacterial strainsthat may become pathogenic, recombinant commensal bacteria, such asStreptococcus sp. or Lactococcus sp., may be used as vaccine carriers.

A potential problem with recombinant commensal microorganisms is thatthey may colonize the mucosal surfaces and generate a long term exposureto the target antigens expressed and released by the recombinantmicroorganisms which may cause immune tolerance.

Additionally, the use of genetically modified microorganisms thatcontain recombinant nucleic acid has met considerable opposition fromthe public as a whole, stemming from a low level acceptance of productswhich contain recombinant DNA or RNA. Similar objections exist againsteven the use of attenuated pathogenic strains or against proteins, orparts of proteins, derived from pathogenic strains. Further, theheterologous surface display of proteins described herein entails theuse of anchoring or targeting proteins specific and selective for alimited set of microorganisms, which are of recombinant or pathogenicnature which greatly restricts their potential applications.

The protein anchor of L. lactis, AcmA (cA), its homologs and functionalderivatives (PCT International Patent Publication No. W099/25836) bindin a non-covalent manner to a wide variety of Gram-positive bacteria.Binding also occurs to isolated cell-wall material. The ligand to whichthe protein anchor of L. lactis binds in these cell walls is currentlyunknown.

The use of a gram-positive, food-grade bacterium, such as Lactococcuslactis, offers significant advantages over the use of other bacteria,such as Salmonella, as a vaccine delivery vehicle. For instance, L.lactis does not replicate in or invade human tissues and reportedlypossesses low intrinsic immunity (Norton et al. 1994). Further, mucosaldelivered L. lactis that expresses tetanus toxin fragment C has beenshown to induce antibodies that protect mice against a lethal challengewith tetanus toxin even if the carrier bacteria was killed prior toadministration (Robinson et al. 1997). The killed bacteria still containrecombinant DNA that will be spread into the environment, especiallywhen used in wide-scale oral-immunization programs. However, theuncontrollable shedding of recombinant DNA into the environment may havethe risk of being taken up by other bacteria or other microorganisms.

SUMMARY OF THE INVENTION

The invention discloses a method for improving binding of aproteinaceous substance to cell-wall material of a Gram-positivebacterium. The proteinaceous substance comprises at least one repeat,but may comprise two or three repeat sequences of an AcmA cell-wallbinding domain, homolog or functional derivative thereof. The methodcomprises treating the cell-wall material with a solution capable ofremoving a cell-wall component, such as a protein, lipoteichoic acid orcarbohydrate, from the cell-wall material and contacting theproteinaceous substance with the treated cell-wall material. Improvedbinding may be obtained by treating the cell-wall material with asolution capable of removing a cell-wall component. The cell-wallmaterial may be subsequently stored until it is contacted with a desiredfusion protein. The fusion protein may comprise an AcmA cell-wallbinding domain, homolog or functional derivative thereof where thecell-wall material is contacted with the fusion protein. The method ofthe present invention may be used to obtain cell-wall material with animproved capacity for binding a proteinaceous substance comprising theAcmA cell-wall binding domain, homolog or functional derivative thereof.

The invention also discloses a method for removing components from abacterial cell-wall comprising treating whole cells with a solutioncapable of removing a cell-wall component such as a protein,lipoteichoic acid or carbohydrate from the cell-wall material. Thecell-wall material obtained by the present invention yields cell-wallmaterial with at least 20%, better 30%, best 40% or even 50% ofrelatively empty, but intact, cell envelopes which include inertspherical microparticles. The inert spherical microparticles will bereferred to herein as bacterial “ghosts.” The tenn “ghosts” reflects thesize and shape of the bacterium from which the ghosts are obtained.

The invention also discloses a method for obtaining cell-wall materialof a Gram-positive bacterium with an improved capacity for binding witha proteinaceous substance comprising an AcmA cell-wall binding domain,homolog or functional derivative thereof The method comprises treatingthe cell-wall material with a solution capable of removing a cell-wallcomponent such as a protein, lipoteichoic acid or carbohydrate from thecell-wall material, wherein the cell-wall material comprises sphericalpeptidoglycan microparticles referred to herein as ghosts.

Methods to extract bacterial cell-wall material with a solution havebeen described in EP 0 545 352 A and Brown et al. (Prep. Biochem. 6:479,1976). A method to obtain purified soluble peptidoglycan from bacteriaby exposure to TCA has been disclosed. The cited references describeprocedures in which cells are mechanically disrupted, wherein theresulting cell fragments are treated with TCA to extract peptidoglycansfrom the cell-wall. The cited methods provide a peptidoglycanpreparation and a lysed, randomly fragmented cell-wall preparation fromwhich cell-wall components have been removed. However, these methods donot yield ghosts. Furthermore, the methods do not allow targeting with aproteinaceous substance comprising an AcmA cell-wall binding domain,homolog or functional derivative thereof.

The method of the present invention is aimed at yielding ghosts fromwhich cell-wall components have been removed. The use of ghosts fordisplay of proteinaceous substances has advantages over the use of thedisrupted cell-wall material. For example, binding the proteinaceoussubstance to bacterial ghosts results in a higher packing density whencompared to binding a substance to mechanically disrupted cell-wallmaterial. A high density surface display of proteins is favorable forapplication in industrial processes. In one embodiment, the presentinvention discloses a method for obtaining the cell-wall material notinvolving rupture.

Cell-wall material obtained by mechanical disruption methods suffersfrom several practical drawbacks. Because cells are completely brokenwith mechanical disruption, intracellular materials are released fromthe cell and cell-wall fragments need to be separated from a complexmixture of proteins, nucleic acids, and other cellular components. Thereleased nucleic acids may increase the viscosity of the solution andcomplicate processing steps, especially chromatography. The cell debrisproduced by mechanical lysis also often includes small cell fragmentswhich are difficult to remove. These problems are overcome when ghostsare produced using methods of the present invention. The uniformcomposition of a ghost preparation including particle size and shapeoffers other advantages for subsequent purification and isolation steps.The invention thus discloses a method of obtaining cell-wall materialnot involving rupture of the cell-wall, wherein the resulting cell-wallmaterial comprises ghosts.

The use of bacterial ghosts is often preferable when compared to the useof mechanically disrupted cell-wall bacteria for the surface display ofimmunogenic determinants. In contrast to mechanical disruptionprocedures, ghosts are produced by a process that preserves most of thebacteria's native spherical structure. Bacterial ghosts are better ableto bind to and/or are more easily taken up by specific cells or tissuesthan mechanically disrupted cell-wall material. The ability of bacterialghosts to target macrophages or dendritic cells enhances theirfunctional efficacy. Thus, the non-recombinant, non-living ghost systemdisclosed by the present invention is well suited as a vaccine deliveryvehicle. Accordingly, the invention discloses a method for obtainingghosts, wherein the ghosts have an improved capacity for binding with aproteinaceous substance and have an enhanced induction of the cellularimmune response.

The invention also discloses a method for binding a proteinaceoussubstance to the cell-wall material of a Gram-positive bacterium,wherein the proteinaceous substance comprises an AcmA cell-wall bindingdomain, homolog or functional derivative thereof. The method comprisestreating the cell-wall material with a solution capable of removing acell-wall component such as a protein, lipoteichoic acid or carbohydratefrom the cell-wall material, and subsequently contacting theproteinaceous substance with the cell-wall material. The cell-wallmaterial comprises ghosts which have been produced by the presentinvention which does not involve rupture of the bacterial cell-wall.

In another embodiment, the solution capable of removing the cell-wallmaterial has a pH that is lower than the calculated Pi value of the AcmAcell-wall binding domain, homolog or functional derivative thereof.Particularly, the solution comprises an acid such as acetic acid (HAc),hydrochloric acid (HCl), sulphuric acid (H₂SO₄), trichloro acetic acid(TCA), trifluoro acetic acid (TFA), and monochloro acetic acid (MCA).The concentration of the acid in the solution will be dependent on thedesired pH value which may be determined by calculation using computerprogram such as DNA star or Clone Manager. For instance, when thecalculated pI is >8, pH values of about 6 to 4 may suffice for effectingappropriate binding. When pI values are calculated to be lower, such asaround 6, pH values of 3–4 may be selected. When domains with calculatedpI values ranging from 8 to 12 are encountered, using the solutioncomprising 0.06 to 1.2 M TCA, or comparable acid, may suffice.

The binding may be improved by heating the cell-wall material or ghostsin the solution. However, precise requirements for the heating may varydepending on the cell-wall material or ghosts. However, heating for 5–25minutes at approximately boiling temperature (i.e., 100° C.) will oftengenerate the desired cell-wall material with improved binding capacity.The cell-wall material may then be washed and pelleted (e.g., bycentrifugation) from the treatment solution and subsequently be stored(e.g., by freezing) or freeze-drying until further use. Such cell-wallmaterial includes spherical peptidoglycan microparticles that usuallyreflect the size and shape of the bacterium from which they wereobtained.

In one embodiment, the cell-wall material is derived from a Lactococcus,a Lactobacillus, a Bacillus or a Mycobacterium sp. The cell walls ofGram-positive bacteria include complex networks of peptidoglycan layers,proteins, lipoteichoic acids and other modified carbohydrates.Generally, chemical treatment of the cell-wall material may be used toremove cell-wall components such as proteins, lipoteichoic acids andcarbohydrates, wherein the chemical treatment yields purifiedpeptidoglycan (Morata de Ambrosini et al. 1998). Sodium dodecyl sulphate(SDS) is also commonly used to remove proteins. Trichloro acetic acid(TCA) is known to specifically remove lipoteichoic acids andcarbohydrates from cell-wall isolates. Phenol, formamide and mixtures ofchloroform and methanol are other examples of organic solvents that maybe used to enhance the purification of peptidoglycan.

In the present invention, the effect of the pretreatment of whole cellsof gram-positive bacteria with these and other chemicals in relation tobinding technology provides the possibility to obtain bacterial ghostsor cell-wall material derived from the bacteria which possess new traits(i.e., different binding properties) without the introduction ofrecombinant DNA.

In another embodiment, the present invention discloses the incorporationof cell-wall material with improved binding capacity for AcmA-typeanchors into a composition, such as pharmaceutical composition, with aproteinaceous substance comprising an AcmA-type anchor. Reactive groups,such as antigenic determinants, heterologous enzymes, single-chainantibodies, polyhistidyl tags, peptides, and other compounds may bebound to the cell-wall material as disclosed herein by providingreactive groups with an AcmA-type anchor, and subsequently contactingthe cell-wall material with the reactive groups to improve bindingcapacity. Other reactive groups include fluorescing protein, luciferase,binding protein or peptide, antibiotics, hormones, non-peptide antigenicdeterminants, carbohydrates, fatty acids, aromatic substances orreporter molecules.

In another embodiment, the invention discloses the use of cell-wallmaterial in generating bioadsorbents or biofilters for environmentalpurposes, microbiocatalysts, and diagnostic tools. For instance, the useof immobilized biocatalysts, such as enzymes or whole microbial cells,has increased steadily during the past decade in the food,pharmaceutical and chemical industries. The immobilized biocatalysts aremore stable, easier to handle, and can be used repeatedly in industrialprocesses in comparison to their free counterparts. Immobilization ofenzymes typically requires a chemical step to link the enzyme to aninsoluble support. However, chemical treatments may negatively affectthe enzymes. Alternatively, enzymes may be immobilized by incorporationin gels with the disadvantage-that diffusion of the substrate into thegel slows down the process.

As disclosed herein, large-scale immobilization of enzymatically activeproteins may be accomplished by surface displaying proteins ongram-positive cells or cell-wall material. For instance, theimmobilization of a fusion protein comprising α-amylase or β-lactamasefused to the AcmA-protein anchor domain has been demonstrated herein inL. lactis. The addition of the AcmA-anchor fusion protein resulted inthe stable attachment of heterologous proteins to the surface of L.lactis and other gram-positive bacteria. Further, pretreating L. lactiscells and other gram-positive cells with acid as described hereinresults in a high density surface display of heterologous proteins andis a prerequisite for application in industrial processes. Further, thecarrier or gram-positive cells may be obtained in high yield and benon-recombinant. Thus, a method disclosed herein may be used toeconomically produce the immobilized enzyme and make the AcmA-proteinanchor a useful approach for the surface display of enzymes ongram-positive cells.

Another industrial application of an immobilized enzyme is theisomerization of glucose which is catalyzed by glucose isomerase andused during the production of high-fructose corn syrup. This process maybe made economically feasible by immobilizing the glucose isomerase. Theproductivity of glucose isomerase is improved by increasing thestability of epoxide hydrolase in organic solvents by immobilization tomicrobial cells or cell-wall material as described herein.

Immobilized enzymes may also be used to treat waste water or industrialeffluent. For instance, industrial effluents containing low valuechemicals produced during synthesis of the commodity chemicalsepichlorohydrin and propylene oxide may be treated by using immobilizedhaloalkane dehalogenase to recycle these low value products into themanufacturing process.

The invention further discloses chimeric or hybrid AcmA-type anchors forthe preparation of a composition that has new binding properties. TheAcmA-type anchors can be divided into two groups of hybrids based ontheir pI (see, table 3). A large group includes hybrids with a pI higherthan 8, but lower than 10, and a smaller group includes hybrids with arelatively low pI (i.e., <5). Hybrid AcmA type anchors are disclosedwith at least one AcmA type domain and a relatively high calculated pI,and another AcmA type domain is disclosed with a relatively lowercalculated pI. The resulting hybrid anchor has an intermediatecalculated pI which is useful when release of the bound proteinaceoussubstance at a higher pH is contemplated. Such a composition may berouted through the stomach, which has relative low pH, such that thecomposition releases its anchor bound reactive groups in the intestineswhich have a higher pH.

The invention also discloses a proteinaceous substance comprising anAcmA cell-wall binding domain, homolog or functional derivative thereofwherein the binding domain is a hybrid of at least two differentAcmA-type cell-wall binding domains, homologs or functional derivativesthereof. The proteinaceous substance may comprise an AcmA cell-wallbinding domain, homolog or functional derivative thereof where thebinding domain is a hybrid of at least two different AcmA repeatsequences and has a calculated pI lower than 10. For instance, a hybridprotein anchor including the A1 and A2 repeat sequences of AcmA and theD1 repeat sequence of AcmD may be constructed. Such a hybrid domain maycomprise at least one AcmA type domain with a relatively high calculatedpI and another AcmA type domain with a relatively lower calculated pI.The domain with the relatively high pI may be derived from, orfunctionally equivalent to, the AcmA type domain of the lactococcalcell-wall hydrolase AcmA. Of course, many other domains with a high pIare known, such as those disclosed in table 3. A domain with arelatively low pI may be derived from, or functionally equivalent to,the AcmA type domain of the lactococcal cell-wall hydrolase AcmD.However, other domains with relatively low pI are known, including thosedisclosed in table 3.

The invention further discloses a proteinaceous substance comprising ahybrid domain with at least two stretches of amino acids, wherein eachstretch corresponds to a domain repeat sequence and is located adjacentto each other. The stretches may be separated by one or more amino acidresidues of a short distance, i.e., 3–6 to 10–15 amino acids apart, by amedium distance, i.e., 15–100 amino acids apart, or by longer distances,i.e., >100 amino acid residues apart.

In another embodiment, the invention discloses a proteinaceous substancewith a hybrid AcmA domain that further comprises a reactive group.Reactive groups that may be used include, without limitation, antigenicdeterminants, heterologous enzymes, single-chain antibodies or fragmentsthereof, polyhistidyl tags, fluorescing proteins, luciferase, bindingproteins or peptides, antibiotics, hormones, non-peptide antigenicdeterminants, carbohydrates, fatty acids, aromatic substances, inorganicparticles such as latex, and reporter molecules. The reactive group mayalso include AcmA cell-wall binding domains, homologs or functionalderivatives thereof wherein the binding domain is a hybrid of at leasttwo different AcmA cell-wall binding domains, homologs or functionalderivatives thereof that are useful in heterologous surface display andare broadly reactive with cell-wall components of a broad range ofmicro-organisms. As used herein, the AcmA cell-wall binding domains,homologs and functional derivitives thereof will also be referred to ashybrid AcmA domains.

The invention further discloses reactive groups which are non-proteinmoieties, including substances such as antibiotics, hormones, aromaticsubstances, inorganic particles, or reporter molecules. The substancesmay be constructed by binding an antibiotic, such as penicillin,tetracycline or various other antibiotics, a hormone, such as a steroidhormone, or any other compound to a binding domain produced by thepresent invention. Such binding may be achieved using various techniquesknown in the art and may function to label or “flag” the binding domain.For instance, a binding domain may be bound to a reporter molecule suchas fluorescent nanoparticles, i.e., FITC or HRPO, wherein tools aregenerated that may be used in diagnostic assays to detect microorganismspossessing peptidoglycan. Similarly, a binding domain may be bound to anantibiotic and used for in vivo parenteral administration into thebloodstream of humans or animals, or used in vitro to bindmicroorganisms with peptidoglycan in order to increase the concentrationof the antibiotic around the microorganism which may be killed by theantibiotics.

The invention further discloses a reactive group which is a proteinmoiety which may include, without limitation, antigenic determinants,enzymes, single-chain antibodies or fragments thereof, polyhistidyltags, fluorescing proteins, binding proteins or peptides. For instance,a protein including a reactive group which is another protein orpolypeptide is disclosed. The invention also discloses a nucleic acidmolecule encoding the protein produced using the methods of theinvention. Such a nucleic acid molecule, comprising single-stranded ordouble-stranded DNA, RNA or DNA-RNA duplex, comprises nucleic acidsequences which encode a hybrid binding domain. The nucleic acidmolecule may also comprise nucleic acid sequences encoding the reactivegroup polypeptide and may further comprise other nucleic acid sequencesencoding a signal peptide comprising promoter sequences or regulatorynucleic acid sequences.

A vector comprising a nucleic acid molecule encoding a proteinaceoussubstance provided by the invention is also disclosed. Examples ofvectors include, without limitation, a plasmid, a phage or a virus,wherein the vectors may be constructed using nucleic acids of theinvention and routine skills known in the art. Viral vectors includebaculovirus vectors or comparable vector viruses through which a proteinproduced by the present invention may be expressed or produced in cells,such as insect cells.

A host cell or expression system including a nucleic acid molecule or avector produced using methods of the present invention are alsodisclosed. The host cell expressing a protein of the present inventionmay be a microorganism to which the protein is attached. The host cell,or expression system, may be a Gram-positive bacterium, a Gram-negativebacterium, a yeast cell, an insect cell, a plant cell, a mammalian cell,or a cell-free expression system, such as a reticulocyte lysate. Thehost cell or expression system may be constructed or obtained using anucleic acid or vector of the present invention and routine skills knownin the art.

In a further embodiment, the invention discloses a pharmaceuticalcomposition comprising cell-wall material with an improved bindingcapacity with an immunogen bound thereto which may be useful forvaccination purposes, i.e., a vaccine. The vaccine may be used to invokeimmunity against pathogens, such as malaria, which undergo life cyclestages where the pathogen is not in the blood, but hides in cells.

The vaccines may be delivered to mucosal surfaces instead of beinginjected since mucosal surface vaccines are easier and safer toadminister. A L. lactis derived cell-wall material may be used formucosal vaccination since this bacterium is of intestinal origin and noadverse immune reactions are generally expected from L. lactis.

The vaccine of the invention may also be administered by injection. Whenthe vaccine is administered through injection, cell-wall material may bederived from a Mycobacterium sp. since mycobacterial cell-wallpreparations have beneficial adjuvant properties. The mycobacterialcell-wall vaccine may be mixed with the proteinaceous substance carryingthe immunogenic determinants used in the vaccine.

A vaccine produced using a method of the present invention will likelyhave a reduced risk of generating undesirable immune responses againstcell-wall compounds of unwanted immunogens because the unwantedimmunogens are not included in the vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic map of plasmid pNG3041 that encodes the reporterprotein MSA2::cA that is secreted as a proprotein using the lactococcalPrtP signal- and prosequences (PrtP.sspro). Pnis represents the nisininducible promoter of the nisA gene. T represents the transcriptionalterminator. CmR is the chloramphenicol resistance gene. repC and repAare genes involved in the replication of the plasmid.

FIG. 2A. Fluorescence microscopic images of bacterial cells withexternally bound MSA2::cA. Lb. curvatis, Lb. sake and L. lactis cellsthat were not pre-treated prior to binding.

FIG. 2B. Fluorescence microscopic images of bacterial cells withexternally bound MSA2::cA. L. lactis cells that were TCA pretreatedprior to binding. The light colored areas indicate the position wherethe reporter protein MSA2::cA binds. The difference between L. lactiscells that were not pretreated with TCA (in FIG. 2A) and those therewere TCA pretreated is apparent (in FIG. 2B).

FIG. 3. Western blots of chemically pretreated L. lactis cells that werewashed after the pretreatment and incubated with MSA2::cA to allowbinding. Unbound MSA2::cA was removed by washing. The drawing shows theMSA2::cA that was bound to the chemically pretreated cells and detectedusing an antibody specific for MSA2. The different pretreatments areindicated above the lanes. MSA2::cA is produced by the producer cells asa proprotein, pro-MSA2::cA. Some pro-MSA2::cA is present in the mediumused for binding and binds as indicated by the arrow. A membrane boundprotease, HtrA, of the producer cells cleaves off the pro-sequenceresulting in mature MSA2::cA, which also binds to the pretreated cellsas indicated by the asterisk. HtrA also cleaves off the repeats of thecA anchor. Since there are three repeats, MSA2 proteins of several sizesare present in the medium of the producer. As long as more than onerepeat is present, binding can still occur. The double asterisks pointto MSA2::cA from which one or two repeats have been cleaved. M is amolecular weight marker. The molecular weights are indicated in the leftmargin. The two blots have different signal intensities. As a reference,both blots contain the same TCA-pretreated samples. The difference insignal intensity is due to differences in stain developing time. It isapparent that the TCA and other acid pretreatments produce pronouncedeffects on the subsequent binding of MSA2::cA. The conclusions for allchemical pre-treatments are summarized in Table 1.

FIG. 4. Coomassie stained SDS-PAGE gel with chemically pre-treated L.lactis cells. Pre-treatments: (1) no-treatment; (2) HCl; (3) H₂SO₄; (4)HAc; (5) TFA; and (6) TCA. It is apparent that treatment of the cellswith HCl, H₂SO₄, TFA or TCA significantly removes an amount of proteinfrom the cells.

FIG. 5. Western blot of L. lactis cells TCA pre-treated with differentTCA concentrations and externally bound with MSA2::cA. Arrow andasterisks: as in FIG. 3. Pretreatments: (1) no TCA-treatment; (2) 1%TCA; (3) 5% TCA; (4) 10% TCA; and (5) 20% TCA. An increase in thebinding of MSA2::cA is shown to correlate with increasing amounts of TCAused in the pretreatment.

FIG. 6. Alignment of cA repeats with cD repeats. AcmA (A1) (SEQ ID NO:16) is aligned to AcmD (D1) (SEQ ID NO: 19). AcmA (A2) (SEQ ID NO: 17)is aligned to AcmD (D2) (SEQ ID NO: 20). AcmA (A3) (SEQ ID NO: 18) isaligned to AcmD (D3) (SEQ ID NO: 21). Consensus repeats SEQ ID NO: 163,164 and 165 are aligned. The amino acids that are in agreement with theconsensus sequence are shown at the bottom of the figure (defined in PCTPublication WO99/25836) are underlined. The asterisks indicate residuesthat are identical between the compared repeats.

FIG. 7. Binding of various anchor-fusion proteins to L. Lactis with andwithout TCA pretreatment. Multiple bands shown in one lane are caused bythe different processed forms of MSA2 fusions. Lanes: (1) non-pretreatedL. lactis+MSA2::cA; (2) non-treated L. lactis+MSA2::cD; (3)non-pretreated L. lactis+MSA2; (4) TCA-pretreated L. lactis+MSA2::cA;(5) TCA-pretreated L. lactis+MSA2::cD; and (6) TCA-pretreated L.lactis+MSA2. The effect of TCA pretreatment on the binding of MSA2::cAis shown (i.e., compare lanes 1 and 4). A minor improvement for MSA2::cDand no improvement for MSA2 without anchor is observed. Since there is asignal for MSA2 without the anchor means that MSA2 by itself has a weakaffinity for bacterial cell walls. However, MSA2::cD or MSA2 binding tothe pretreated cells cannot be detected using fluorescence or electronmicroscopy (see text). The difference in results is probably due to adifference in sensitivity of the techniques

FIG. 8. Fluorescence microscopy image of TCA-pretreated L. lactis cellsincubated with MSA2::cA or MSA2::cD. Light colored areas indicate theposition were the reporter fusion protein binds. It appears that bindingonly occurred with MSA2::cA and not with MSA2::cD.

FIG. 9A. Electron microscopy images of L. lactis cells incubated withdifferent MSA2 constructs. The black dots represent the position ofbound MSA2 fusion protein. Image A depicts non-pretreated cellsincubated with MSA2::cA.

FIG. 9B. Electron microscopy images of L. lactis cells incubated withdifferent MSA2 constructs. The black dots represent the position ofbound MSA2 fusion protein. Image B depicts TCA-pretreated cellsincubated with MSA2::cA. Significant binding, shown by black dots, isonly visible in the TCA-pretreated cells incubated with MSA2::cA.

FIG. 9C. Electron microscopy images of L. lactis cells incubated withdifferent MSA2 constructs. The black dots represent the position ofbound MSA2 fusion protein. Image C depicts TCA-pretreated cellsincubated with MSA2::cD.

FIG. 9D. Electron microscopy images of L. lactis cells incubated withdifferent MSA2 constructs. The black dots represent the position ofbound MSA2 fusion protein. Image D TCA-pretreated cells incubated withMSA2.

FIG. 10. Binding of different anchor-fusion proteins to B. subtilis withand without TCA pretreatment. The drawing is a Western blot similar toFIGS. 3 and 7. Lanes: (1) non-pretreated cells +MSA2::cA; (2)non-pretreated cells+MSA2::cD; (3) non-pretreated cells+MSA2; (4)TCA-pretreated cells+MSA2::cA; (5) TCA-pretreated cells+MSA2::cD; (6)TCA-pretreated cells+MSA2; and (7) non-pretreated B. subtilis (negativecontrol). TCA pretreatment improves the binding of MSA2::cA in a mannersimilar as L. lactis (i.e., compare lanes 1 and 4). Only backgroundbinding is observed for MSA2::cD and MSA2 without anchor.

FIG. 11. Fluorescence microscopy image of MSA2::cA binding to Lb. caseiwith or without TCA pretreatment. The light colored areas representbound MSA2::cA. TCA pretreatment improves binding of MSA2::cA and Lb.casei.

FIG. 12. Fluorescence microscopy image of MSA2::cA and MSA2::cD bindingto M. smegmatis pretreated with TCA. The light colored areas representbound MSA2 fusion protein. As illustrated, only MSA2::cA binds.

FIG. 13. Western blot of L. lactis cells with externally bound MSA2::cAtreated with LiCl or stored under different conditions. The bands in thevarious lanes represent the amount of MSA2::cA that remained bound tothe TCA pretreated cells. Arrow and asterisks: as in FIG. 3. Lanes: (1)marker; (2) non-pretreated L. lactis incubated without MSA2::cA; (3)non-pretreated L. lactis incubated with MSA2::cA; (4) TCA-pretreated L.lactis incubated with MSA2::cA; (5) TCA-pretreated L. lactis incubatedwith MSA2::cA, subsequently washed with 8 M LiCl.; (6) TCA-pretreated L.lactis incubated with MSA2::cA, subsequently stored in water for 3 weeksat 4° C.; (7) TCA-pretreated L. lactis incubated with MSA2::cA,subsequently stored in 10% glycerol for 3 weeks at −80° C.; and (8)TCA-pretreated L. lactis incubated with MSA2::cA, subsequently stored inwater for three weeks at −80° C. As illustrated, TCA pretreatmentimproves binding of MSA2::cA to L. lactis cells (i.e., compare lanes 3and 4). Washing with 8 M LiCl and storage in water for 3 weeks at 4° C.has minor effects on the bound MSA2::cA (i.e., compare lane 4 with 5 and6). Storage at −80° C. has no effect on the bound MSA2::cA (i.e.,compare lane 4 with 7 and 8).

FIG. 14A. Fluorescence microscopy image of MSA2::cA surface expressionin the recombinant strain NZ9000(pNG3041). The light colored areasindicate the position of MSA2 fusion protein. The recombinant strainproducing MSA2::cA has the protein on the surface in some specificspots.

FIG. 14B. Fluorescence microscopy image of MSA2::cP surface expressionin the recombinant strain NZ9000(pNG3043). The recombinant strainproducing MSA2::cP has more on the surface organized in several areas.

FIG. 14C. Fluorescence microscopy image of MSA2::cA binding to TCApretreated L. lactis cells. The surface of the TCA-pretreatednon-recombinant L. lactis with bound MSA2::cA is completely covered withthe protein.

FIG. 15. Western blots of L. lactis total protein extracts reacted withrabbit immune serum diluted at 1:100. 0: preimmune serum. 2 and 3: serumafter the second and third immunization, respectively. A1:subcutaneously immunized rabbit with NZ9000ΔacmA(pNG3041) cells(recombinant, MSA2::cA surface anchored). B1: subcutaneously immunizedrabbit with NZ9000ΔacmA (negative control). C2: orally immunized rabbitwith NZ9000ΔacmA(pNG3043) cells (recombinant, MSA2::cP surfaceanchored). E1: orally immunized rabbit with TCA-pretreated NZ9000ΔacmAto which MSA2::cA had been externally bound (non-recombinant, MSA2::cAsurface anchored). The staining bands in the lanes illustrates that L.lactis proteins react with the indicated rabbit antiserum. It is visiblethat the non-recombinant TCA-pretreated strain with bound MSA2::cA (E1)evokes a minimal response to L. lactis proteins indicating that theresponse to the carrier is reduced, while the response to the malariaantigen is not negatively influenced (see, Table 2).

FIG. 16. Schematic representation of the domains in AcmA and AcmD. SSrepresents signal sequence. Both enzymes include a cell-wall bindingdomain that includes 3 repeats indicated by A1, 2, 3 and D1, 2, 3. Thealignments of these repeats are shown in FIG. 6. In addition, an exampleof one of the hybrid protein anchors is described in Table 5.

FIG. 17. Western blot showing the effect of pH supernatant on binding ofMSA2::cD to TCA-pretreated L. lactis cells. As previously described, theWestern blot shows the amount of MSA2::cD bound by the cells. Inaddition, the amount of MSA2::cD that was not bound and remained in themedium after binding is shown. The arrow indicates the expected positionfor pro-MSA2::cD and the asterisk indicates the position of matureMSA2::cD. Lanes: (1) pH during binding 6.2, cells; (2) pH during binding6.2, supernatant after binding; (3) pH during binding 3.2, cells; (4) pHduring binding 3.2, supernatant after binding; and (5) positive control:L. lactis, TCA-pretreated with bound MSA2::cA at pH6.2. It is visiblethat MSA2::cD binds better at pH 3.2 than at pH 6.2 (i.e., compare lanes1 and 3).

FIG. 18. Western blot of medium supernatant (S) after binding to ghostcells at the indicated pH's and ghost (G) with the bound protein anchor.Lanes 1 and 2 illustrate binding at pH 3; lanes 3 and 4 illustratebinding at pH 5; lanes 5 and 6 illustrate binding at pH 7. The drawingshows considerable binding at pH 5. At pH 5, the native cD anchor(D1D2D3) shows little binding. The addition of the A3 repeat, which hasa high pI value, results in increased binding at pH 5.

FIG. 19. Immunization schedule. Mice immunizations were started at day 1and repeated after 14 and 28 days. A lethal nasal challenge with S.pneumoniae was given 14 days after the last oral immunization. S.c.represents subcutaneous immunization.

FIG. 20. Serum antibody response. Mean anti-PpmA serum antibody titers.OV represents orally immunized; IN represents intranasally immunized; SCrepresents subcutaneously immunized; Freunds PpmA refers to soluble PpmAsubcutaneously administrated with Freunds complete adjuvants. Hightiters were obtained with the intranasally and subcutaneouslyadministrated Ghosts-PpmA::cA.

FIG. 21. Survival times. The orally vaccinated mice were challenged witha lethal dose of S. pneumonia. Mice vacinnated with soluble PpmA orGhost alone died within 72 hours. Forty percent of the mice immunizedwith Ghosts-PpmA::cA survived the challenge, indicating they wereprotected by the vaccination.

DETAILED DESCRIPTION EXAMPLE 1

Acid Pretreatment of Gram-Positive Bacteria Enhances Binding of AcmAProtein Anchor Fusions.

Materials and Methods.

Bacterial Strains And Growth Conditions. Lactococcus lactis strainMG1363 (Gasson 1983) or derivatives thereof, such as MG1363ΔacmA (Buistet al. 1995) or NZ9000Δ acmA, were used as recipients for binding ofreporter fusion protein. NZ9000 (Kuipers et al. 1997) which carries oneof the reporter plasmids was used as a production strain. L. lactisstrains were grown in M17 broth (Oxoid) supplemented with 0.5% glucosein standing cultures at 30° C. Chloramphenicol was added to the M17medium to an end-concentration of 5 μg/ml when appropriate. Forexpression, mid-log phase cultures were induced for 2 hours with theculture supernatant of the nisin producing L. lactis strain NZ9700 asdescribed by Kuipers et al. (1997). Lactobacillus casei, ATCC393, wasgrown in MRS broth (Oxoid) in standing cultures at 30° C. Mycobacteriumsmegmatis, ATCC700084, was grown in Middlebrook medium (Oxoid) at 37° C.in aerated cultures. Bacillus subtilis, 168, was grown in TY broth (perliter: 10 g tryptone, 5 g yeast extract, 5 g NaCl pH7.4) at 37° C. inaerated cultures.

Construction Of Reporter Plasmids. The merozoite surface antigen 2(MSA2) of Plasmodium falciparum strain 3D7 (Ramasamy et al. 1999) fusedto the three repeats of AcmA (MSA2::cA) was used as the reporter anchorprotein. The reporter anchor protein is encoded by plasmid pNG3041 basedon the nisin inducible expression vector pNZ8048 (Kuipers et al. 1997)and contains a modified multiple cloning site in which the hybridreporter gene was cloned. An in frame fusion of the reporter was made atthe 5′ end the lactococcal PrtP signal—and prosequence, and at the 3′end the AcmA protein anchor sequence. The sequence of the MSA2 gene thatwas included in the construct corresponds to nucleotides (nt) 61 to 708in Genbank accession number A06129. Primers used for the amplificationof the MSA2 gene were MSA2.1 (5′-ACCATGGCAAAAAATGAAAGTAAATATAGC (SEQ IDNO:2)) and MSA2.4 (5′-CGGTCTCTAGCTTATAAGCTTAGAATTCGGGATGTTGCTGCTCC ACAG(SEQ ID NO:3)). The primers contain tags with restriction endonucleaserecognition sites that were used for cloning. For cloning of the PrtPsignal and prosequence (nt 1206 to 1766 in Kok et al. 1988), the primersPrtP.sspro.fw (5′-CCGTCTCCCATGCAAAGGAAAAAAGA AAGGGC (SEQ ID NO:4)) andPrtP.sspro.rev (AAAAAAAGCTTGAATTCCCAT GGCAGTCGGATAATAAACTTTCGCC (SEQ IDNO:5)) were used. The primers include restriction sites that were usedfor cloning. The AcmA protein anchor gene fragment (nt 833 to 1875) wasobtained by subcloning a PvuII-HindIII fragment from plasmid pAL01(Buist et al. 1995). Restriction endonuclease enzymes and Expand HighFidelity PCR polymerase were used in accordance with the instructions ofthe supplier (Roche). The final expression vector was designated pNG3041(FIG. 1).

A construct including a stopcodon introduced after the MSA2 sequence inpNG3041 was designated pNG304. The protein secreted using this constructis substantially the same as the protein expressed from the pNG3041plasmid except that the protein produced from pNG304 does not containthe AcmA protein anchor. The protein produced from pNG304 is used as anegative control in the binding assays. A vector was also made in whichthe AcmA protein anchor was exchanged for a protein anchor. The putativecell-wall binding domain of L. lactis AcmD (Bolotin et al. 2001) wascloned (nt 1796 to 2371 in Genbank accesssion number AE006288) usingprimers pACMB2 (5′-CGCAAGCTTCTGCAGAGCTCTTAGATTCTAATT GTTTGTCCTGG (SEQ IDNO:6)) and pACMB3 (5′-CGGAATTCAAGGAGGAGAAATA TCAGGAGG (SEQ ID NO:7)) toproduce the plasmid pNG3042. pNG3042 contains an in-frame fusion betweenMSA2 and the protein anchor of AcmD (MSA2::cD) and differs from plasmidpNG3041 only in the gene fragment encoding the protein anchor.

Cell Pretreatment and Binding Conditions. Chemical pretreatment of L.lactis NZ9000ÄacmA was done with 10% TCA (0.6 M) in the followingmanner. Cells of 0.5 ml stationary phase cultures were sedimented bycentrifugation and washed once with 2 volumes demineralized water. Cellswere resuspended in 1 volume of a 10% TCA solution and incubated byplacing the reaction tube in boiling water for 15 minutes. Subsequently,cells were washed once with 2 volumes PBS (58 mM Na₂HPO₄.2H₂O, 17 mMNaH₂PO₄.H₂O, 68 mM NaCl; pH 7.2) and three times with 2 volumesdemineralized water. The cells were used directly for bindingexperiments or stored (as described herein) until further use.

The following chemicals and conditions were used to examine the effectof different chemicals on the binding capacity of L. lactis cells forAcmA-type protein anchor fusions: acetic acid (HAc), hydrochloric acid(HCl), sulfuric acid (H₂SO₄), TCA, and trifluoroacetic acid (TFA),monochloro acetic acid (MCA). The acids were used at a finalconcentration of 0.6 M and incubated for 15 minutes in boiling water.SDS, dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO) were used ata concentration of 10%. The SDS pretreatment was incubated for 15minutes in boiling water and DMF and DMSO treatments were incubated atroom temperature for 15 minutes. Cells were also pretreated with phenol(Tris buffer saturated) and incubated for 15 minutes at 55° C. Otherchemicals pretreated at the 55° C. incubation temperature were: 4 Mguanidine hydrochloride (GnHCl), 37% formaldehyde, chloroform:methanol(CHCL₃:CH₃0H (2:1)) and 0.1% sodium, hypochlorite (NaOCl). In addition,incubation with 25 mM dithiothrietol (DTT) for 30 minutes at 37° C. anda pretreatment with hexane (100%) were analyzed.

The effect of enzymatic pretreatment of cells with lysozyme was alsotested. For lysozyme pretreatment, the cells were resuspended in buffer(20% sucrose, 10 mM Tris pH 8.1, 10 mM EDTA, 50 mM NaCl) with lysozyme(2 mg/ml) and incubated at 55° C. for 15 minutes. After the chemical andenzymatic pretreatments, the washing steps were the same as the washingsteps used for the TCA treated cells. TCA pretreatment of Bacillussubtilis, Lactobacillus casei and Mycobacterium smegmatis was done asdescribed herein for L. lactis.

Cell-free culture supernatants containing MSA2::cA, MSA2::cD or MSA2without anchor were incubated in four-fold excess for 10 minutes at roomtemperature with pretreated cells (e.g., cells from 0.5 ml culture wereincubated with 2.0 ml culture supernatant). After binding, cells weresedimented by centrifugation, washed twice in 2 volumes demineralizedwater, resuspended in SDS-denaturation buffer, heated for 5 minutes at98° C., subjected to SDS-PAGE, and analyzed by Western blot analysis.

Storage Conditions. Cell-free supernatants containing MSA2::cA, MSA2::cDor MSA2 were stored at −20° C. with or without 10% glycerol prior tobinding. TCA pretreated L. lactis cells were stored at −80° C. in 10%glycerol prior to binding. TCA pretreated L. lactis cells with boundMSA2::cA were stored at +4° C. or −80° C. with or without 10% glycerol.Cells stored in 10% glycerol were washed once with 1 volume ofdemineralized water prior to binding.

Cell pellets (in demineralized water) of TCA pretreated L. lactis cellswith or without bound MSA2::cA were frozen by contacting the vials withliquid nitrogen and removing the water with lyophilization.Alternatively, non-frozen cell pellets were dried under vacuum at 30° C.for 2 hours prior to binding.

Western Blotting. For detection of MSA2 proteins, cell pelletscorresponding to 500 μl culture were resuspended in 50 μlSDS-denaturation buffer. Cell-free culture supernatants (1 ml) wereconcentrated by phenol-ether precipitation (Sauve et al. 1995), vacuumdried and resuspended in 50 μl SDS-denaturation buffer. Proteins wereseparated with standard SDS-PAGE techniques. After separation, proteinswere electroblotted onto PVDF membranes (Roche). In immunoblots, MSA2proteins were detected with 1:10,000 diluted rabbit MSA2-specificantiserum (Ramasamy et al. 1999) and 1:5,000 diluted anti-rabbitIgG-conjugated alkaline phosphatase (Roche) using known procedures.

Fluorescence Microscopy. 100 μl cell suspensions incubated withMSA2::cA, MSA2::cD or MSA2 fusion proteins were washed twice withdemineralized water and resuspended in an equal volume PBS containing 1%BSA and MSA2-specific rabbit antiserum diluted to 1:200. Afterincubation for 20 minutes at room temperature, the cells were washedthree times with 2 volumes PBS. Subsequently, the cells were incubatedfor 20 minutes in 1 volume PBS with 1% BSA and 1:100 diluted Oregongreen labeled goat anti-rabbit immunoglobulin G (Molecular Probes).After washing once with 2 volumes PBS and twice with 2 volumesdemineralized water, the cells were resuspended in 100 μl demineralizedwater. A 10 μl aliquot of the resuspended cells was spread onto aPolysin microslide (Menzel-Gläser), air dried, and examined under afluorescence microscope (Zeiss).

Electron microscopy. TCA-pretreated L. lactis cells incubated withMSA2::cA, MSA2::cD or MSA2 were collected and washed as describedherein. Immunogold labeling was performed on whole mount preparations ofglutaraldehyde fixed cells on Formvar-carbon coated nickel grids usingAuroprobe 15 nm goat anti-rabbit IgG gold marker (Amersham). Primaryantibodies against MSA2 were diluted 1:1000 in PBS-glycine buffer. Thelabeled samples were stained with 0.1% uranyl acetate (W/V in water) andexamined in a Philips CM10 transmission electron microscope at 100 kV.

Pretreatment of L. Lactis Cells With Different Chemicals. The cA proteinanchor of L. lactis AcmA can be used to bind fusion proteins to a widevariety of Gram-positive bacteria. However, the amount of fusion proteinthat binds varies greatly among this group of bacteria. Binding ofMSA2::cA that covers the entire cell surface of some lactobacilli wasobserved, whereas other bacteria such as L. lactis showed only limitedlocalized binding (FIG. 2A). This phenomenon may be due to the fact thatthe cell walls of some bacterial species contain components thatinterfere with cA anchor binding. Since chemicals like SDS, TCA,chloroform/methanol and others may be used to remove components fromisolated bacterial cell walls (Morata de Ambrosini et al. 1998), theeffect of the removal of cell-wall components from L. lactis whole cellson the binding of the reporter fusion protein MSA2::cA was investigated.L. lactis cells were pretreated as described herein with variouschemicals or with lysozyme.

FIG. 3 shows typical Western blots of pretreated whole cells to whichMSA2::cA was bound. Mature MSA2::cA migrates at a position of a 75 kDaprotein (indicated by an asterisk). The arrow represents MSA2::cA thatcontains the PrtP prosequence. The double asterisks represent MSA2::cAfrom which one or two of the repeats have been removed. A cell membraneanchored protease HtrA has been shown to be involved in processingproproteins and in removing repeats from AcmA (Poquet et al. 2000). Fromthe results of FIG. 3, it may be concluded that pretreatment with TCA(lanes 8 and 16 contain the same samples, the difference in signalintensity is due to differences in stain developing time), HCl, H₂SO₄and HAc substantially improves the subsequent binding of MSA2::cA(compare with the negative control in lane 15). Other tested acids, TFAand MCA, had similar effects (not shown). Phenol, GnHCl, formamide andchloroform-/methanol pretreatments showed a moderate improvement ofbinding (lanes 4, 5, 6, 7, respectively). Minor binding improvementswere observed after pretreatment with SDS, DMF, DMSO and DTT. Theresults are summarized in Table 1. Based on the results, it appears thatpretreatment of L. lactis cells with the acids TCA, TFA, MCA, HCl, H₂SO₄and HAc are the most effective agents for improving binding of cA anchorfusion proteins to lactococcal cells. Acids such as TCA are known toremove lipoteichoic acids from cell walls.

Whether proteins are removed from the cell walls by these acidtreatments was also analyzed. FIG. 4 shows a Coomassie stained gel oflysed pretreated cells. Most of the acid treatments, except for HAc,removed a substantial amount of proteins from the lactococcal cells.Since HAc removed only trace amount of proteins (compare lane 1 and 4)and SDS pretreatment (which is known to remove proteins from the cellwalls) showed only a minor improvement of MSA2::cA binding (FIG. 3, lane1), it may be concluded that removal of proteins from the cell wall isnot critical for improving the binding of cA anchor fusions. Thisconclusion may be due to the fact that lipoteichoic acids orcarbohydrates occupy sites in the cell walls of L. lactis that interferewith efficient binding. Alternatively, acid pretreatment may result inaltering the compactness of peptidoglycan strands that make cA bindingsites more available.

TCA pretreatment was also used in all other experiments. The optimal TCAconcentration in the boiling procedure was determined. TCA percentagesof 1, 5, 10 and 20% were tested. Although 1% TCA pretreatment alreadyshowed a significant improvement in binding of MSA2::cA and 5% TCApretreatment showed a further increase, no further improvement wasobserved at concentrations higher than 10% TCA (FIG. 5). Therefore, theboiling procedure with 10% TCA was selected as the standard procedurefor the experiments.

The binding characteristics of the lactococcal cA homolog cD in a MSA2fusion were analyzed using the standard TCA pretreatment procedure. Twoof the three AcmD repeats are highly homologous to those of AcmA. Analignment is shown in FIG. 6. Secreted MSA2 without an anchoring domainwas included in these experiments as a negative control. In Westernblots, the effect of TCA pretreatment on the binding of MSA2::cA wasevident (FIG. 7, compare lanes 1 and 4). The effect of TCA pretreatmentwas also studied using fluorescence microscopy (FIG. 2, compare L.lactis in A and B; FIG. 8) and electron microscopy (FIG. 9, compare Aand B). Independent of the technique used, the effect of TCApretreatment on the binding of MSA2::cA can be detected.

The binding of MSA2::cD to non-TCA pretreated L. lactis cells was low asdetected in Western blots (FIG. 7, lane 2) and was undetectable influorescence microscopy and electron microscopy (FIG. 9A). TCApretreatment only had minor effects on the intensity of the MSA2::cDsignal in Western blots (FIG. 7, lane 5). At the same time, no MSA2::cDspecific signal associated with the pretreated cells could be observedin fluorescence microscopy (FIG. 8) and only low levels of labeling wasobserved in electron microscopy (FIG. 9C). Some cell-associated signalwas observed for MSA2 without anchoring domain for both non-TCApretreated and TCA pretreated L. lactis cells (FIG. 7, lanes 3 and 6,respectively). However, for MSA2::cD, this was not observed influorescence microscopy (not shown) and only minor labeling signals werefound in electron microscopy (FIG. 9D). Taken together, it may beconcluded that: (i) the reporter protein MSA2 does have some low degreeof affinity for bacterial cell walls that can be detected in Westernblots; (ii) the cA anchor domain specifically stimulates the binding ofthe reporter fusion to non-pretreated cells; (iii) chemicalpretreatment, especially with acids, enhances this binding; and (iv) thecD anchor domain does not promote binding of fusion proteins under theconditions applied.

The fluorescence microscopic images and electron microscopic images ofTCA pretreated lactococcal cells (FIGS. 2, 8 and 9) showed thatpretreatment leaves the integrity of the cell intact. However, cells areno longer viable (plating efficiency 0) and therefore may be consideredas inert spherical peptidoglycan microparticles with a diameter ofapproximately 1 μm, “ghost cells”.

Binding to Other Gram-Positives. The binding of MSA2::cA, MSA2::cD andMSA2 without anchor domain to the Gram-positive bacteria B. subtilis,Lb. casei and M. smegmatis were also analyzed. FIG. 10 shows a Westernblot summarizing binding of MSA2::cA, MSA2::cD and MSA2 tonon-pretreated and TCA-pretreated B. subtilis cells. As for L. lactis,an increase in binding is observed for MSA2::cA. A MSA::cA specificsignal could also be visualized in fluorescence microscopy ofnon-pretreated B. subtilis cells, but with a highly improved signal forthe TCA-pretreated cells (not shown). Binding of MSA2::cD and MSA2 tonon-pretreated or TCA-pretreated cells could not be demonstrated influorescence microscopy (not shown).

Similar results were obtained for Lb. casei and M. smegmatis. Theimproved binding of MSA2::cA to TCA-pretreated Lb. casei cells is shownin FIG. 11. For MSA2::cD and MSA2, no fluorescence signals were detected(not shown). The TCA-pretreatment of M. smegmatis also had a positiveeffect on the binding of MSA2::cA, whereas no binding was observed forMSA2::cD or MSA2 (FIG. 12). Taken together, it may be concluded thatacid pretreatment, such as with TCA, improves the binding of cA proteinanchor fusions to the cell surface of Gram-positive bacteria.

Binding strength and storage conditions. The strength of the MSA2::cAbinding to TCA-pretreated L. lactis cells was analyzed with a treatmentof LiCl after the binding. LiCl is commonly used to remove proteins frombacterial cell walls. From the Western blot of FIG. 13, it may beconcluded that 8 M LiCl partially removes MSA2::cA from the L. lactiscells (compare lanes 4 and 5). Therefore, although MSA2::cA bindsnon-covalently to cell walls, the binding interactions are most likelyvery strong.

Cell-free culture supernatants with MSA2::cA were stored with or without10% glycerol at −20° C. MSA2::cA stored in this manner for several weekshad the same capacity to bind to TCA-pretreated L. lactis cells (notshown).

TCA-pretreated L. lactis cells with bound MSA2::cA were stored for 3weeks at +4° C. in demineralized water or at −80° C. in demineralizedwater with or without 10% glycerol. The samples were analyzed in Westernblots. Storing pretreated cells with bound MSA2::cA for 3 weeks in waterat +4° C. only resulted in a loss of signal of about 50% (FIG. 13,compare lanes 4 and 6). Whether this loss of signal was due todegradation or due to release of the protein into the water was notdetermined. Storage at −80° C. with or without 10% glycerol had noeffect on the binding (FIG. 13, compare lanes 4, 7 and 8).

In addition, the effects of drying and lyophilization on the binding ofMSA2::cA to TCA-pretreated L. lactis cells were studied. Drying ofpretreated cells had no observable negative effect on binding ofMSA2::cA afterwards. Dried pretreated cells with bound MSA2::cA could beresuspended in water without losing bound fusion protein. This was alsoobserved for lyophilized cells with bound MSA2::cA. Lyophilization ofTCA-pretreated cells prior to binding resulted in loss of the bindingcapacity for MSA2::cA (results not shown).

From these data, it may be concluded that: (i) in spite of thenon-covalent character of cA anchor binding to cell walls, the bindingis very strong; (ii) cell-free culture supernatants can be stored safelyat −20° C.; and (iii) drying of TCA-pretreated cells provides anefficient and simple method for storage of such cells either with orwithout bound cA-anchor fusions.

EXAMPLE 2

Oral Immunizations of Rabbits with Non-recombinant Lactococcus lactisPreloaded with the Plasmodium falciparum Malaria Antigen MSA2 Fused tothe Lactococcal AcmA Protein Anchor.

In Example 1, a technology is described that efficiently binds proteinhybrids when externally added to the cell surface of non-recombinantgram-positive bacteria by means of an AcmA-type protein anchor. Thistechnology provides the possibility to provide bacteria or bacterialcell walls with new traits without introducing recombinant DNA intothem. The immunogenicity in rabbits of the Plasmodium falciparummerozoite surface protein, MSA2 of strain 3D7 (Ramasamy et al. 1999),presented on the cell surface of non-recombinant non-living L. lactiscells as an AcmA anchor fusion protein was investigated.

Materials and Methods.

Bacterial Strains and Growth Conditions. The L. lactis strain whichproduces MSA2::cA, the strain's growth conditions, the induction forexpression, the TCA pretreatment of the L. lactis recipient cells andthe binding of MSA2::cA to the cells was described in example 1 with thefollowing modification: a ratio of 1 (TCA-pretreated cells) to 5(cell-free culture supernatant with MSA2::cA) was used for binding. AnL. lactis NZ9000 strain carrying plasmid pNG3043 was used as a positivecontrol in the immunization experiments (was positive in a previousunpublished experiment). Plasmid pNG3043 encodes an MSA2 hybrid proteinthat contains the lactococcal PrtP cell-wall anchoring domain at itsC-terminus (MSA2::cP) instead of the AcmA protein anchor. The PrtPcell-wall anchoring domain contains the LPXTG (SEQ ID NO: 1) motif thatenables a membrane-linked sortase to covalently couple the protein tothe cell wall (Navarre and Schneewind 1994). The cP domain used inconstruct pNG3043 corresponds to nt 6539 to 6914 in Kok et al. (1988).Primers used for the amplification of this fragment were PrtP.cwa.fw3(5′-ATATAAAGCTTGCAAAGTCTGAAAACGAAGG (SEQ ID NO: 8)) and PrtP.cwa.rev(5′-CCGTCTCAAGCTCACTATTCTTCACGTTGTTTCCG (SEQ ID NO: 9)). The primersinclude restriction endonuclease recognition sites for cloning. PlasmidpNG3043 differs from plasmid pNG3041 in the cell-wall binding domain.Growth conditions and induction of expression of strainNZ9000ΔacmA(pNG3043) were the same as for strain NZ9000 ΔacmA(pNG3041).

Rabbit Immunizations. Ten barrier-reared, New Zealand white rabbitsobtained from Harlan laboratories, The Netherlands, were used in groupsof 2 for experimental immunizations. The care and use of animals wereaccording to WHO guidelines (WHO/LAB/88.1). The rabbits were ear bledprior to immunization to obtain preimmune sera. Details of the rabbitsand immunogens are as follows:

Rabbits A1 and A2 were subcutaneously immunized withNZ9000ΔacmA(pNG3041) cells (recombinant, MSA2::cA partly surfaceanchored).

Rabbits B1 and B2 were subcutaneously immunized with NZ9000ΔacmA(negative control).

Rabbits C1 and C2 were orally immunized with NZ9000ΔacmA(pNG3043) cells(recombinant, MSA2::cP surface anchored).

Rabbits D1 and D2 were orally immunized with NZ9000ΔacmA(pNG3041) cells(recombinant, MSA2::cA surface anchored).

Rabbits E1 and E2 were orally immunized with TCA treated NZ9000ΔacmA towhich MSA2::cA had been bound from NZ9000ΔacmA(pNG3041) culturesupernatant (non-recombinant, MSA2::cA surface anchored).

Stocks of NZ9000ΔacmA(pNG3043) with MSA2::cP expressed at its surfacewere stored in aliquots of 10¹¹ cells in growth medium containing 10%glycerol at −80° C. The cells remain viable under these conditions andretain MSA2 on the surface as demonstrated by immunofluorescence (notshown). The first immunization was carried out with freshly grownbacteria. For subsequent immunizations, stocks of bacteria were freshlythawed, washed and resuspended in buffer at the appropriateconcentration for immunizations.

On the other hand, the non-pretreated NZ9000ΔacmA (negative control),the non-pretreated NZ9000ΔacmA(pNG3041) and the TCA-pretreatedNZ9000ΔacmA with the externally bound MSA2::cA were prepared daily fromfresh cultures.

Subcutaneous injections were performed with a total of 5×10⁹ cells in100 μl PBS without any adjuvant into two sides on either side of thespine. The subcutaneous injections were repeated two more times at 3week intervals. Prior to oral immunization, the rabbits were deprived ofwater and food for 2–4 hours. The rabbits were then fed 5×10¹⁰ cellsresuspended in 1 ml of 0.5% sucrose. Each dose was repeated for threesuccessive days to obtain reproducible oral immunization. Altogether,three series of oral immunizations were given at 3 week intervals.Adverse effects consequent to the immunizations, including granulomas atthe sites of subcutaneous injections, were not observed indicating thatL. lactis was well tolerated by the animals.

Serum Antibody Responses. Rabbits were ear bled 2 weeks after eachimmunization to obtain sera for antibody assays. The sera were stored at−20° C. until use. Ten-fold serial dilutions of the antisera in 2% BSAin PBS were used in immunofluorescence assays (IFA) to determine thetiter of the antibodies against MSA2 on the surface of 3D7 P. falciparummerozoites. IFA was performed on acetone-methanol fixed late stage 3D7P. falciparum parasites as previously described (Ramasamy 1987). Fordetection of antibody isotypes, Oregon Green conjugated goat anti-rabbitIg (Molecular Probes) was used as the second antibody. For detection ofIgG antibodies, a fluorescein conjugated, affinity purified, mousemonoclonal with specificity against rabbit IgG chains (Rockland) wasused.

Results and Discussion.

Surface Expression of MSA2 in Different L. Lactis Strains. Coomassiestaining of SDS-PAGE gels and fluorescence microscopy were used todetermine, in a semi-quantitative way, the number of MSA2 moleculesexpressed and surface exposed by the recombinant lactococcal strainscarrying plasmid pNG3041 or pNG3043 that produce MSA2::cA or MSA2::cP,respectively, and by the non-recombinant TCA-pretreated L. lactis cellsto which MSA2::cA had been bound from the outside. The recombinantstrains were estimated to produce approximately 1.4×10⁵ molecules ofMSA2::cA or MSA2::cP. The surface exposure of MSA2::cA and MSA2::cPdiffered considerably as shown by fluorescence microscopy in FIG. 14.The non-recombinant TCA-pretreated L. lactis cells with bound MSA2::cAshowed a uniform staining of the entire cell surface. However, thesemi-quantitative SDS-PAGE analysis indicated that about 1×10⁴ moleculesof MSA2::cA per cell were represented.

Accordingly, it may be concluded that the number of surface exposedMSA2::cA and MSA2::cP on the recombinant lactococcal strains is lessthan 10% of the total number of molecules produced by these strains. Theother molecules are most likely trapped in the membrane or the cellwall. Similar observations were made by Norton et al. (1996) for theexpression of TTFC fused to the cP cell-wall anchoring domain. In thatstudy, only membrane-associated or cell-wall-associated TTFC could bedemonstrated and no surface-exposed TTFC::cP was demonstrated. Thus, itappears that binding from the outside to TCA-pretreated cells is a moreefficient method to surface-expose proteins on L. lactis cells.

Anti-MSA2 Antibody Responses In Orally Immunized Rabbits.Characteristics of the anti-MSA2 antibody response to the immunizationsare summarized in Table 2. The oral immunizations with the recombinantL. lactis that produces MSA2::cP (rabbits C1 and C2) were done before(unpublished results) and used as a positive control. In the previousexperiment, a similar antibody response was found. The presentexperiment showed that specific antibodies against near native MSA2 weredetectable after two immunizations for group A, D and E rabbits, andthat antibody titers increased in all instances after a thirdimmunization. IgG antibodies were predominant after three immunizationsin either the subcutaneous or oral route. A comparatively weak anti-MSA2surface IFA, attributable to the generation of cross-reactive antibodies(as described herein), was also observed after three controlsubcutaneous immunizations with L. lactis cells alone.

Taken together, the results indicate that: (i) MSA2 produced bylactococcal cells elicits serum antibodies that recognize native P.falciparum parasite MSA2; (ii) MSA2-specific T_(h) cells are activatedthrough mucosal immunization due to the presence of systemic IgGantibodies (Table 2) that can be boosted (unpublished results); and(iii) oral immunizations with MSA2::cA bound to non-recombinantnon-living TCA-pretreated L. lactis cells are as efficient in evokingspecific serum antibody responses as the live recombinant strainproducing MSA2::cA that was administered subcutaneously or orally, or asefficient as the live recombinant strain producing MSA2::cP that bindsMSA2 covalently to its cell wall delivered orally.

Anti-Lactococcal Antibody Responses. Western blots (FIG. 15)demonstrated significant antibody responses against L. lactis antigensafter two and three immunizations of the rabbits. The responses werenotably greater after subcutaneous (group A and B rabbits) than oralimmunization with L. lactis (group C rabbits). Oral immunization withthe TCA-pretreated lactococcal cells (group E rabbits) elicitedantibodies that reacted at a lower intensity with fewer L. lactisantigens than oral immunization with viable L. lactis cells. This ismost likely due to the fact that proteins are removed from thelactococcal cells by the TCA pretreatment (see, example 1). The loweranti-carrier response observed for the TCA-pretreated (non-recombinant)cells renders this type of delivery vehicle more suitable for repeatedimmunization strategies than its untreated (recombinant) counterpart.

EXAMPLE 3

pH-Dependent Cell-Wall Binding of AcmA Protein Anchor Homologs andHybrids.

The cell-wall binding domain or anchor of the lactococcal cell-wallhydrolase AcmA includes three repeats of 45 amino acids that show a highdegree of homology (Buist et al. 1995). These three repeats belong to afamily of domains that meet the consensus criteria as defined in PCTpublication WO 99/25836 and can be found in various surface locatedproteins in a wide variety of organisms. Another feature that most ofthese domains have in common is that their calculated pI values arehigh, approximately 8 or higher (Table 3). The pH used in previousbinding experiments with MSA2::cA (i.e., examples 1 and 2) wasapproximately 6, indicating that the binding domain was positivelycharged.

The AcmA protein anchor homolog of the lactococcal cell-wall hydrolaseAcmD (cD) (Bolotin et al. 2001) also includes three repeats (FIG. 16)with a calculated pI that is lower (approximately pI 3.8) than that ofthe cA domain (Table 4). Consequently, the cD anchor was negativelycharged at the binding conditions used in example 1. No binding of theMSA2::cD reporter protein occurred under these conditions asdemonstrated herein. Therefore, the influence of the pH during bindingof a cD fusion protein (MSA2::cD) was investigated. Furthermore, ahybrid protein anchor including the three cD repeats and one cA repeatthat has a calculated pI value that is higher than that of the cDrepeats alone was constructed. The hybrid protein anchor showed betterbinding pH values above the pI of the cD repeats alone, indicating thatthe pH binding range of AcmA-type protein anchors can be manipulated byusing the pI values of the individual repeats in hybrids.

Materials and Methods.

Bacterial strains, growth and induction conditions, TCA pretreatment ofL. lactis cells, incubation of the MSA2 protein anchor fusion proteinsto TCA-pretreated cells, washing conditions, protein gelelectrophoresis, Western blotting and immunodetection were the same asdescribed herein with reference to example 1. The cell-free culturesupernatants with MSA2::cA, MSA2::cD or A3D1D2D3 have a pH ofapproximately 6.2. The influence of pH was examined by adjusting the pHof the cultures by the addition of HCl or NaOH to obtain the requiredpH.

Plasmid Constructions. The plasmid that expresses the MSA2::cD fusionwas described herein with reference to example 1. Plasmid pPA43 is basedon the same expression plasmid and contains an in frame fusion of thelactococcal signal sequence of Usp45 (ssUsp; van Asseldonk et al. 1990.Gene 95: 155–160), the c-myc epitope for detection purposes, the A3 cArepeat and repeats D1, D2 and D3 of cD. Primers used for cloning A3 werecArepeat3.fw (CCG TCT CCA ATT CAA TCT GCT GCT GCT TCA AAT CC (SEQ ID NO:10)) and cA repeat3.rev (TAA TAA GCT TAA AGG TCT CCA ATT CCT TTT ATT CGTAGA TAC TGA CCA ATT AAA ATA G (SEQ ID NO: 11)) (the primers include theA3 specific sequences). The primers used for cloning the three cDrepeats were cDrepeat1.fw (CCGTCTCCAATTTCAGGAGGAACTGCTGTTACAACTAG) (SEQID NO: 12) and cDrepeat3.rev(TAATAAGCTTAAAGGTCTCCAATTCCAGCAACTTGCAAAACTTCTCCT AC) (SEQ ID NO: 13)(the primers include the cD specific sequences).

Results and Discussion.

Binding of MSA2::cD at Low pH. Since binding of MSA2::cD was notobserved at a pH (the pH of the culture medium after growth andinduction is about 6.2) higher than the calculated pI for the cD domain(i.e., pI 3.85), binding was studied when the pH of the medium wasadjusted to pH 3.2. TCA-pretreated L. lactis cells were used as thebinding substrate and the relative amounts of bound MSA2::cD wereanalyzed in Western blots. The amounts of unbound reporter proteinremaining in the culture supernatant after binding were also analyzed.FIG. 17 shows a clear increase in bound MSA2::cD when binding isperformed at pH 3.2 (compare lanes 1 and 3). At the same time, lessunbound reporter protein remained in the supernatant (compare lanes 2and 4). This result indicates that positive charges are important forbinding of cA-type anchoring domains.

Binding of cAcD Hybrid Anchors. Analysis of the pI values of the cAhomologs in Table 3 indicates that two classes of repeats can bedistinguished: a majority (99 out of 148) of homologs that have a highpI value (>8) and a smaller group (33 out 148), of which cD is arepresentative, that has pI values lower than 6. Based on theexperimental results, it is shown that these types of anchoring domainsonly bind to bacterial cell walls at a pH that is lower than theanchoring domains pI. Notably, most cell-wall binding domain homologsinclude repeats with a pI that are representatives of one of the twogroups, i.e., only repeats with a high or low pI. Some proteins withcell-wall binding domains, e.g., those of DniR of Trepanoma pallidum andan amidase of Borrelia burgdorferi, include repeats with high and lowpI. Since the binding pH of such “natural hybrid” cell-wall bindingdomains is below the intermediate pI value of the total number ofrepeats present in the domain, a hybrid cell-wall protein anchor wasconstructed using the cA and cD repeats with an intermediate pI value.Table 5 lists the native AcmA and AcmD anchors and a number of examplesof cA/cD hybrids. The constructed hybrid protein anchor (A3 D1D2D3) hasa calculated pI value of approximately 5.1. A protein anchor includingonly D1D2D3 shows little binding at a pH above its calculated pI (asdescribed herein). The A3 (pI 10) domain shows similar binding at pH 5and pH 7.

The binding of the hybrid anchor A3D1D2D3 was tested at pH 3, pH 5 andpH 7. At pH 3, most protein had been bound to the ghost cells (FIG. 18).At pH 5, there was considerable binding (+/−40%), whereas there was onlyminimal binding at pH 7 (+/−20%). This result indicates the pH range ofbinding for eD repeats was shifted to higher pH values by the additionof one cA repeat (A3) that caused a shift in calculated pI values of 3.8to 5.1. The increase of binding at pH 5 for the A3D1D2D3 hybrid cannotbe attributed to binding of the A3 repeat alone. If this was the case,then the same level of binding should occur at pH 7 since the A repeatsshow the same binding at these pH values. In addition, the increasedbinding at pH 5 is not an additive effect in the sense that an extrabinding domain results in increased binding. It has previously beenshown that addition of one repeat to the cA anchor did not result inincreased binding. The binding at the higher pH values of the A3D1D2D3repeats as compared to the D1D2D3 repeats alone, thus may be attributedto the increase in the calculated pI value of the hybrid cA/cD anchor.This demonstrates that pH binding properties of these types of proteinanchors may be manipulated on the basis of the pI values of individualrepeats present in the hybrid anchor.

EXAMPLE 4

Induction of Cellular Immune Responses in Mice after Oral Immunizationswith Lactococcal Ghosts Displaying the Malaria Plasmodium falciparumAntigen MSA2 Fused to the Lactococcal AcmA Protein Anchor.

Non-genetically modified non-living Lactococcus lactis cells (ghosts)preloaded with the Plasmodium falciparum MSA2 antigen fused to the AcmAprotein anchor (MSA2::cA) were used to orally immunize mice in a similarway as described herein with reference to example 2. In this experiment,the question of whether immunizations through the oral route with thenon-recombinant non-living Ghosts carrying MSA2::cA on their surface(Ghosts-MSA2::cA) can elicit typical Th1-type immune responses, such asIgG2 antibodies and gamma-interferon (γIFN) producing T cells in thespleen is addressed. These responses are particularly relevant to obtainimmunity for pathogens, such as malaria, that undergo stages in theirlife cycle where they are not in the blood, but hide in cells.

Materials and Methods.

Groups of five mice of different strains were used for immunization. Thestrains of mice used were Balb/c (with the major histocompatibilitylocus allotype of H2d), C57 Black (H2b), C3H (H2k) and ICR (out bred,i.e., of varying H2 types). Oral immunizations were performed at threeweekly intervals. Immunizations were performed with MSA2::cA absorbed onto the surfaces of TCA treated Lactococcus lactis cells(Ghosts-MSA2::cA) or with recombinant L. lactis that displayed MSA2 onthe surface through the use of a covalently linked cell-wall anchor (L.lactis(MSA2::cP)) as described herein with reference to example 2. Themice were tail bled to obtain serum samples two weeks after the second,third and fourth immunizations. Fecal pellets were collected andextracted to examine intestinal IgA antibody production. The mice weresacrificed at the end of each experiment and the spleens were removedfor examining T-cell responses by ELISPOT. MSA2-his tag produced in E.coli was used as antigen in the ELISA and ELISPOT assay. The growth ofbacterial strains and the preparation of Ghost cells was as describedherein with reference to example 2.

Results and Discussion

Kinetics and Isotypes of the Serum IgG Antibodies Generated OralImmunizations. Differences in the kinetics of the antibody response andthe isotype distribution were observed between different murine strains.The antibody response was also different when living recombinant L.lactis (MSA2::cP) or Ghosts-MSA2::cA were used as immunogens. WithGhosts-MSA2::cA, high serum antibody levels were detectable in the C3Hmice after two immunizations. IgG antibodies were detectable in all fourmurine strains after three and four immunizations. Antibody titers werehighest in C3H mice. IgG antibodies that reacted with native MSA2 onparasites were detected in the sera of immune mice by fluorescencemicroscopy (IFA) confirming that the immunizing form of the proteinelicits biologically relevant antibodies. Control immunizations wereperformed with Ghosts alone where no MSA2-specific antibodies wereelicited. In parallel experiments using MSA2cP as the immunogen, highserum IgG antibody levels were only seen with Balb/c mice after twoimmunizations. After three and four immunizations, good antibodyresponses developed in C3H mice. Antibody titers were highest in Balb/cmice.

Significant differences existed between the strains in the isotypes ofthe elicited serum IgG antibodies in response to immunization withGhosts-MSA2::cA. Balb/c mice showed higher levels of IgG2a and IgG2bantibodies, some IgG3 antibodies and negligible IgG1 which demonstratesa possible Th1 bias. On the other hand, C57 Black and C3H mice had highIgG1, IgG2a and IgG2b, and lower IgG3 antibodies to MSA2 which is morecharacteristic of a mixed Th1 and Th2 response. ICR mice, as expected,showed a range of responses. Some ICR mice had the Balb/c and others theC3H/C57 Black pattern of IgG isotypes.

Formation of Mucosal Antibodies. IgA antibodies were detected by ELISAin the fecal pellets of the ICR and Balb/c mice, but were not detectedin C3H or C57 Black mice when immunization was performed with livingrecombinant L. lactis(MSA2::cP) or Ghost-MSA2::cA.

T-Cell Responses. The increase of the intensity of the IgG ELISAreactions seen in mice immunized with Ghosts-MSA2::cA with eachimmunization demonstrates that boosting takes place and that aTh-dependent antibody response exists in these animals. The IgG isotypedistribution further confirms this conclusion. Therefore, Th cells aregenerated in ICR, Balb/c, C57 Black and C3H mice.

The ELISPOT assay for detecting gamma-interferon (γIFN) producing cellsdetects mainly CD8⁺ Tc cells, which are an important component of theimmune response to many pathogens, including malaria parasites.His-tagged MSA2 produced in E. coli was used as antigen in the assay.MSA2-specific γIFN producing cells could be detected in the spleens ofBalb/c, C57 Black and C3H mice that were immunized with Ghosts-MSA2::cA.MSA2-specific γIFN producing cells were not observed in the spleens ofcontrol mice immunized with Ghosts alone or with the living recombinantL. lactis(MSA2-cP). The latter group showed a high level of non-specificγIFN producing cells. The high background observed may be due to ongoinginflammation.

The sensitization of MSA2-specific Tc cells in the spleen afterimmunization with the non-recombinant non-living L. lactis Ghost-systemcarrying a foreign protein is a novel finding which is applicable tomalaria since protection against sporozoite-infection is associated withγIFN producing cells being produced in the spleen.

The non-recombinant non-living Ghost system can be used in oralimmunizations to elicit typical Th1-type immune responses. These typesof responses are particularly relevant to obtain immunity for pathogensthat undergo stages in their life cycle where the pathogens are not inthe blood, but rather hide in cells. The responses are more pronouncedand more specific for the Ghost system than for the living recombinantsystem. The Ghost system has the additional advantage of eliminating therisk of spreading recombinant DNA into the environment.

EXAMPLE 5

Protection of Mice for Lethal Streptococcus pneumoniae Challenge afterOral Immunizations with Lactococcal Ghosts Preloaded with PpmA AntigenFused to the Lactococcal AcmA Protein Anchor.

Streptococcus pneumoniae is the leading etiological agent of severeinfections including septicemia, meningitis, pneumonia, and otitismedia. Recent studies on the molecular epidemiology and pathogenesis ofS. pneumoniae have identified pneumococcal proteins with vaccinepotential. One of these proteins, the protease maturation protein PpmA,has been shown to elicit immune protective potential in a mousepneumonia model.

The non-genetically modified lactococcal ghosts have been shown to be anefficient carrier for use in oral immunizations of rabbits and mice inorder to elicit strong anti-malaria immune responses. The constructionof lactococcal ghosts that display the S. pneumoniae PpmA fused to thelactococcal AcmA cell-wall binding domain on their surface is describedherein. The ability of these ghosts to protect orally immunized micefrom a lethal nasal dose of S. pneumoniae was investigated.

Materials and Methods.

Bacterial Strains and Growth Conditions. L. lactis was grown and ghostcells were prepared as described herein with reference to example 1. S.pneumoniae was grown as described before (Gingles et al. 2001. InfectImmun 69: 426–434).

Construction of ppmA Protein Anchor Fusion Expression Plasmid. Theexpression plasmid for ppmA protein anchor fusion (PpmA::cA) wassubstantially similar to the expression plasmid for the MSA2 proteinanchor fusion as described herein with reference to example 2. For thesecretion of PpmA::cA, the secretion signal sequence of the Usp45protein (ssUsp) of L. lactis (van Asseldonk et al. 1990. Gene 95:155–160) was used. The PpmA gene was cloned by PCR using primers ppmA.1(CGGTCTCACATGTCGAAAGGGTCAGAAGGTG CAGACC) (SEQ ID NO: 14) and ppmA.2(CGGTCTCGAATTGCTTCGTTTGATGTACTACTG CTTGAG) (SEQ ID NO: 15) resulting inplasmid pPA32 which contains ppmA as an in frame fusion with ssUsp45 andthe protein anchor (ssUsp::ppmA::cA). Expression of the fusion generesults in the secreted product PpmA::cA. The primers include an Eco31Irestriction enzyme recognition site that was used for digestion of thePCR fragment. This restriction digest produced NcoI and EcoRI stickyends which were used for cloning. The primers also included the ppmAsequences. Chromosomal DNA of S. pneumoniae strain D39 was used as atemplate for the PCR reactions.

Preparation of the Vaccine. Three liters of M17 medium with PpmA::cA,obtained after growth and used to induce producer cells for expressionof L. lactis (pPA32), was centrifuged and filter sterilized (0.2 μm) toremove the producer cells. Ghost cells were prepared from 0.5 liter ofL. lactis NZ9000(ΔacmA). After binding, the ghost cells with PpmA::cA(Ghosts-PpmA::cA) were isolated by centrifugation and washed with PBS.The ghost cells were stored in PBS in aliquots of 2.5×10¹⁰ Ghosts/ml at−80° C. Two control groups included: (i) Ghosts without bound PpmA::cA;for the sample preparation the same amounts of ghost cells were used andthe same centrifugation and washing steps were performed, but thebinding step was omitted; and (ii) soluble PpmA was isolated as ahis-tagged fusion.

Mice Immunizations. Groups of 10 mice (CD-1) were used in theimmunizations. Oral doses included 5×10⁹ Ghosts with or without PpmA::cA(50 μg) or 50 μg soluble PpmA in PBS. Nasal doses included 5×10⁸ Ghostswith or without PpmA::cA (5 μg) or 5 μg soluble PpmA. 10⁸Ghosts-PpmA::cA (1 μg) were subcutaneously injected. For intranasalimmunizations, the mice were slightly anesthetized with Isofluorane.

Intranasal Challenge. The groups of orally immunized mice wereintranasally challenged 14 days after the last booster immunization witha dose of 10⁶ colony forming units (CFU) S. pneumoniae D39 as described(Kadioglu et al. 2000 Infect Immun 68: 492–501). Mice were monitoredafter the challenge for visible clinical symptoms for 7 days, at whichpoint the experiment was ended. Mice that were alive after 7 days wereconsidered to have survived the pneumococcal challenge and mice thatbecame moribund during the 7-day period were judged to have reached theendpoint of the assay. The time the animal became moribund was recorded,and the animal was sacrificed by cervical dislocation.

ELISA Analysis. Serum samples were taken from each mouse before theintranasal challenge and stored at −20° C. before use. Microtiter plateswere coated with 100 μg PpmA/ml in 0.05 carbonate buffer. Serial 10-folddilutions of pooled serum of each group were incubated on the plates asdescribed (Gingles et al. 2001, Infect. Immun. 69: 426–434). Anti-mouseimmunoglobulin-horse-radish peroxidase conjugate was used for detectionand the absorbance was measured at 492 nm.

Results and Discussion.

Serum Antibody Response. Mice were immunized orally, nasally andsubcutaneously according to the scheme shown in FIG. 19. Anti-PpmAantibody titers in the blood serum were determined for each group byELISA assays. The results are given in FIG. 20. As expected, ghostsalone administered orally or nasally, OV Ghosts or IN Ghosts,respectively, did not induce anti-PpmA antibodies. Soluble PpmA given bythe nasal route resulted in only a low anti-PpmA antibody titer whichagrees with the general findings that soluble antigens are not veryimmunogenic when given by the mucosal routes. Ghosts-PpmA::cA providedby the oral route (OV PpmA+Ghost) induced only a low level of anti-PpmAserum antibodies. This contrasts the results for the oral immunizationexperiments described herein with reference to examples 2 and 4 withMSA2::cA. However, the contrast may be antigen-type related.

Intranasal administration of Ghosts-PpmA::cA resulted in a high titer ofanti-PpmA antibodies (IN PpmA+Ghosts). A high titer was also obtained bysubcutaneous administration of Ghosts-PpmA::cA. These titers were lowerby a factor of 5 to 10 when compared to soluble PpmA that wassubcutaneously administered and formulated with the strong Freundscomplete adjuvant (Peter Adrian, Erasmus University Rotterdam, TheNetherlands, unpublished results). In addition, the Freunds PpmA vaccinecontained 50 μg PpmA per dose, whereas the intranasally administeredGhosts-PpmA:cA contains only 5 μg/dose and the subcutaneous Ghost-PpmA::cA vaccine contains only 1 μg PpmA/dose. This result demonstrates theadjuvant effect of the ghost cells. Side effects of the orally, nasallyor subcutaneously administrated ghosts were not observed, which is incontrast to the severe side effects that are usually seen with the useof Freunds adjuvants.

The results demonstrate that high titer serum antibodies can be obtainedby the mucosal route of adminstration. These data also show that ghostcells may be safely used in traditionally injected vaccines without sideeffects in order to induce high titer serum antibodies.

Protection Against Challenge. The mice orally immunized with solublePpmA, Ghosts alone or Ghosts-PpmA::cA were challenged 14 days postimmunization with a lethal intranasal dose of S. pneumoniae. The miceimmunized with soluble PpmA or Ghosts alone died within 72 hours afterchallenge. The group immunized with Ghosts-PpmA::cA showed a survivalrate of 40% (FIG. 21). This results shows that mucosal immunization ofmice with Ghosts-PpmA induces protective immunity against a lethal S.pneumoniae challenge. In conclusion, the non-recombinant non-livingGhost system may be used to elicit high titer serum antibodies and themucosal route of administration may be used to obtain protectiveimmunity against a mucosally acquired pathogen.

REFERENCES

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TABLE 1 Effect of different pretreatments of L. lactis on binding ofMSA2::cA. Treatment Signal on Western blot H₂O − 10% TCA (0.6 M) ++++0.6 M HAc ++++ 0.6 M HCl ++++ 0.6 M H₂SO₄ ++++ 0.6 M TFA ++++ 0.6 M MCA++++ Phenol ++ 4 M GnHCl ++ 37% formaldehyde ++ CHCl₃/MeOH ++ 10% SDS +10% DMF + 10% DMSO + 25 mM DTT + 0.1% NaHClO* − Hexane − Lysozyme* −*Lysis of cells occurred during treatment or washing steps.

TABLE 2 MSA2 antibody titres of rabbit serum determined by IFA onPlasmodium falciparum 3D7 asexual blood stage parasites. ImmunisationRabbit 3^(rd) serum Immunogen P 2^(nd) 3^(rd) IgG A1 s.c. L.lactis[MSA2::cA] 0 2 4 4 A2 s.c. L. lactis[MSA2::cA] 0 2 5 5 B1 s.c. L.lactis 0 0 1 n.d. B2 s.c. L. lactis 0 0 1 n.d. C1 oral L.lactis[MSA2::cP] 0 3 5 5 C2 oral L. lactis[MSA2::cP] 0 2 5 5 D1 oral L.lactis[MSA2::cA] 0 2 4 5 D2 oral L. lactis[MSA2::cA] 0 2 4 5 E1 oralTCAL. lactis + MSA2::cA 0 2 5 5 E2 oral TCAL. lactis + MSA2::cA 0 2 5 4L. lactis strain used: NZ9000ΔacmA (lactococcal cells lacking the cellwall hydrolase AcmA). L. lactis[MSA2::cA] and L. lactis[MSA2::cP] arethe recombinant strains with surface expressed MSA2, cell wall anchoredthrough the non-covalent AcmA binding domain (cA) or the covalent PrtPanchoring domain (cP), respectively. TCAL. lactis + MSA2::cA are thenon-recombinant TCA-pretreated lactococcal cells to which MSA2::cA hadbeen bound externally. P: pre-imrnune serum. S.C.: subcutaneousinjection. Titres are expressed as the negative logarithms of the lowestten-fold dilution of sera giving a detectable reaction on the merozoitesurface. The last column represents the IgG fraction of the antibodyresponse. 0: indicates no detectable reaction at a 1:10 dilution of theserum. n.d.: not done.

TABLE 3 AcmA cell wall binding domain homologs and their calculated pIvalues. (the pI values are indicated directly behind the amino acidsequences) Lactococcus *acmA YTVKSGDTLWGISQR SEQ ID NO:16  9.75245–287(33)  437 U1769600 muramidase lactis YGISVAQIQSANNLKSTIIYIGQKLVLT VKVKSGDTLWALSVK SEQ ID NO:17  9.64 321–363(31)YKTSIAQLKSWNHLS SDTIYIGQNLIVS HKVVKGDTLWGLSQK SEQ ID NO:18 10.06 395–437SGSPIASIKAWNHLS SDTILIGQYLRIK *acmD YKVQEGDSLSAIAAQ SEQ ID NO:19  4.15194–237 QGCI25 YGTTVDALVSANSLE NANDIHVGEVLQVA YTVKSGDSLYSIAEQ SEQ IDNO:20  3.78 258–303 YGMTVSSLMSANGIY DVNSMLQVGQVLQVT V YTIQNGDSIYSIATASEQ ID NO:21  4.15 319–361 NGMTADQLAALNGFG INDMIHPGQTIRI ØTuc2009 *lysYVVKQGDTLSGIASN SEQ ID NO:22  6.31 332–375(10)  428 L31364 glycosidaseWGTNWQELARQNSLS (muramidase) NPNMIYAGQVISFT YTVQSGDNLSSIAIL SEQ ID NO:23 3.45 386–428 LGTTVQSLVSMNGIS NPNLIYAGQTLNY Ø-LC3 *lysB YIVKQGDTLSGIASNSEQ ID NO:24  6.31 333–376(10)  429 U04309 muramidase LGTNWQELARQNSLSNPNMIYSGQVISLT YTVQSGDNLSSIARR SEQ ID NO:25  8.79 387–429LGTTVQSLVSMNGIS NPNLIYAGQTLNY Enterococcus *autolysin YTVKSGDTLNKIAAQSEQ ID NO:26  9.74 363–405(25)  671 P37710 muramidase faecalisYGVSVANLRSWNGIS GDLIFVGQKLIVK YTVKSGDTLNKIAAQ SEQ ID NO:27  9.74431–473(25) YGVTVANLRSWNGIS GDLIFVGQKLIVK YTIKSGDTLNKIAAQ SEQ ID NO:28 9.74 499–541(25) YGVSVANLRSWNGIS GDLIFAGQKIIVK YTIKSGDTLNKISAQ SEQ IDNO:29  9.85 567–609(19) FGVSVANLRSWNGIK GDLIFAGQTIIVK HTVKSGDSLWGLSMQSEQ ID NO:30  9.35 629–671 YGISIQKIKQLNGLS GDTIYIGQTLKVG hirae *mur2YTVKSGDSVWGISHS SEQ ID NO:31  9.35 257–299(38)  666 P39046 muramidaseFGITMAQLIEWNNIK NNFIYPGQKLTIK YTVKSGDSVWKIAND SEQ ID NO:32  7.14338–380(33) HGISMNQLIEWNNIK NNFVYPGQQLVVS YTVKAGESVWSVSNK SEQ ID NO:33 9.91 414–456(32) FGISMNQLIQWNNIK NNFIYPGQKLIVK YTVKAGESVWGVANK SEQ IDNO:34  9.64 489–531(33) NGISMNQLIEWNNIK NNFIYPGQKLIVK YTVKAGESVWGVANKSEQ ID NO:35  7.31 565–607(15) HHITMDQLIEWNNIK NNFIYPGQEVIVKYTVKAGESVWGVADS SEQ ID NO:36  7.15 623–665 HGITMNQLIEWNNIK NNFIYPGQQLIVKListeria *P60 VVVEAGDTLWGIAQS SEQ ID NO:37  8.61  30–72(130)  484 P21171adherence monocytogenes KGTTVDAIKKANNLT and invasion TDKIVPGQKLQVNprotein P60 HAVKSGDTIWALSVK SEQ ID NO:38  9.35 203–245 YGVSVQDIMSWNNLSSSSIYVGQKLAIK innocua *P60 VVVEAGDTLWGIAQS SEQ ID NO:39  8.61 30–72(129)  481 Q01836 adherence and KGTTVDAIKKANNLT invasionTDKIVPGQKLQVN protein P60 HNVKSGDTIWALSVK SEQ ID NO:40  8.35 201–243YGVSVQDIMSWNNLS SSSIYVGQKPAIK ivanovii *P60 VVVEAGDTLWGIAQD SEQ ID NO:41 6.35  30–72(125)  524 Q01837 adherence KGTTVDALKKANNLT and invasionSDKIVPGQKLQIT protein P60 YTVKSGDTIWALSSK SEQ ID NO:42  9.37 198–240(73)YGTSVQNIMSWNNLS SSSIYVGQVLAVK YTVKSGDTLSKIATT SEQ ID NO:43  9.89 314–356FGTTVSKIKALNGLN SDNLQVGQVLKVK seeligeri *P60 VVVEAGDTLWGIAQD SEQ IDNO:44  6.35  30–72(127)  523 Q01838 adherence NGTTVDALKKANKLT andinvasion TDKIVPGQKLQVT protein P60 HTVKSGDTIWALSVK SEQ ID NO:45  8.64200–242(77) YGASVQDLMSWNNLS SSSIYVGQNIAVK YTVKSGDTLGKIAST SEQ ID NO:46 9.62 320–362 FGTTVSKIKALNGLT SDNLQVGDVLKVK welshimeri *P60VVVEAGDTLWGIAQS SEQ ID NO:47  8.61  30–72(125)  524 M80348 adherenceKGTTVDALKKANNLT and invasion SDKIVPGQKLQVT protein P60 HTVKSGDTIWALSVKSEQ ID NO:48  9.35 198–240(75) YGASVQDLMSWNNLS SSSIYVGQKIAVKYTVKSGDSLSKIANT SEQ ID NO:49  9.89 316–358 FGTSVSKIKALNNLT SDNLQVGTVLKVKgrayi *P60 VVVASGDTLWGIASK SEQ ID NO:50  9.42  30–72(104)  511 Q01835adherence TGTTVDQLKQLNKLD and invasion SDRIVPGQKLTIK protein P60YKVKSGDTIWALSVK SEQ ID NO:51  9.57 177–219(79) YGVPVQKLIEWNNLSSSSIYVGQTIAVK YKVQNGDSLGKIASL SEQ ID NO:52  8.59 299–342 FKVSVADLTNWNNLNATITIYAGQELSVK Haemophilus *amiB HIVKKGESLGSLSNK SEQ ID NO:53 10.11294–336  432 P44493 N-acetylmura- influenzae YHVKVSDIIKLNQLK moyl-L-RKTLWLNESIKIP alanine amidase HKVTKNQTLYAISRE SEQ ID NO:54 10.49 387–430YNIPVNILLSLNPHL KNGKVITGQKIKLR *yebA YTVTEGDTLKDVLVL SEQ ID NO:55  3.87131–174  475 P44693 homologous to SGLDDSSVQPLIALD endopeptidasePELAHLKAGQQFYWI of Staphylo- coccus lppB YKVNKGDTMFLIAYL SEQ ID NO:56 6.40 147–190  405 P44833 outer AGIDVKELAALNNLS membrane EPNYNLSLGQVLKISlipoprotein somnus lppB YKVRKGDTMFLIAYI SEQ ID NO:57  8.56 120–164  279L10653 outer SGMDIKELATLNNMS membrane EPYHLSIGQVLKIA lipoproteinHelicobacter dniR HVVLPKETLSSIAKR SEQ ID NO:58 10.02 319–361  372AE000654 regulatory pylori YQVSISNIQLANDLK protein DniR DSNIFIHQRLIIRPseudomonas lppB YIVRRGDTLYSIAFR SEQ ID NO:59 10.06  69–113  297 P45682Lipoprotein aeruginosa FGWDWKALAARNGIA PPYTIQVGQAIQFG Putida nlpDYIVKPGDTLFSIAFR SEQ ID NO:60  9.77  44–87  244 Y19122 lipoproteinYGWDYKELAARNGIP APYTIRPGQPIRFS Sinohizobium nlpD IMVRQGDTVTVLARR SEQ IDNO:61 10.27 166–209  512 U81296 Lipoprotein meliloti FGVPEKEILKANGLKSASQVEPGQRLVIP Synechocystis nlpD HQVKEGESLWQISQA SEQ ID NO:62  4.38 87–130  715 D90915 Lipoprotein sp. FQVDAKAIALANSIS TDTELQAGQVLNIPslr0878 HVVKAGETIDSIAAQ SEQ ID NO:63  5.41   4–47  245 D90907Hypothetical YQLVPATLISVNNQL protein SSGQVTPGQTILIP Aquifex nlpDlYKVKKGDSLWKIAKE SEQ ID NO:64 10.08  26–70(24)  349 AE000700 Lipoproteinaeolicus YKTSIGKLLELNPKL KNRKYLRPGEKICLK YRVKRGDSLIKIAKK SEQ ID NO:6510.95  95–137(37) FGVSVKEIKRVNKLK GNRIYVGQKLKIP YRVRRGDTLIKIAKR SEQ IDNO:66 12.11 174–216 FRTSVKEIKRINRLK GNLIRVGQKLKIP Volvox YTIQPGDTFWAIAQRSEQ ID NO:67  9.03  42–85  309 AF058716 chitinase carteriRGTTVDVIQSLNPGV VPTRLQVGQVINVP f. nagariensis YTIQPGDTFWAIAQR SEQ IDNO:68  9.03 106–149 RGTTVDVIQSLNPGV NPARLQVGQVINVP Staphylococcus ProtAHVVKPGDTVNDIAKA SEQ ID NO:69  5.58 431–474  524 A04512 protein A aureusNGTTADKIAADNKLA DKNMIKPGQELVVD lytN YTVKKGDTLSAIALK SEQ ID NO:70 10.03177–220  383 AF106851 autolysin YKTTVSNIQNTNNIA homolog NPNLIFIGQKLKVPColletotrichum cihl HKVKSGESLTTIAEK SEQ ID NO:71  4.76 110–153(31)  230AJ001441 glycoprotein YDTGICNIAKLNNLA DPNFIDLNQDLQIP lindemuthianumYSVVSGDTLTSIAQA SEQ ID NO:72  5.46 185–228 LQITLQSLKDANPGVVPEHLNVGQKLNVP Chlamydophila amiB IVYREGDSLSKIAKK SEQ ID NO:73  9.46159–201  205 AE001659.1 N-Acetylmura- YKLSVTELKKINKLD moyl-L-AlaSDAIYAGQRLCLQ Amidase Pneumoniae CPn0593 YVVQDGDSLWLIAKR SEQ ID NO:7410.01 316–358  362 AE001643 FGIPMDKIIQKNGLN HHRLFPGKVLKLP NlpDVVVKKGDFLERIARA SEQ ID NO:75 10.17 124–166  233 AE001670 MuramidaseNHTTVAKLMQINDLT TTQLKIGQVIKVP YIVQEGDSPWTIALR SEQ ID NO:76  8.64 188–233NHIRLDDLLKMNDLD EYKARRLKPGDQLRI R Chlamydia NlpD VIVKKGDFLERIARS SEQ IDNO:77  9.99 138–180  245 AE001348 Muramidase trachomatis NHTTVSALMQLNDLSSTQLQIGQVLRVP YVVKEGDSPWAIALS SEQ ID NO:78 10.00 200–245 NGIRLDELLKLNGLDEQKARRLRPGDRLRI R papQ HIVKQGETLSKIASK SEQ ID NO:79  9.89 155–197  200AE001330 YNIPVVELKKLNKLN SDTIFTDQRIRLP Prevotella phg HTVRSNESLYDISQQSEQ ID NO:80 10.72 266–309  309 AF017417 hemagglutinin intermediaYGVRLKNIMKANRKI VKRGIKAGDRVVL Leuconostoc lys YTVQSGDTLGAIAAK SEQ IDNO:81  9.23 335–378  432 endolysin oenos Ø10MC YGTTYQKLASLNGIGSPYIIIPGEKLKVS YKVASGDTLSAIASK SEQ ID NO:82  9.68 389–432YGTSVSKLVSLNGLK NANYIYVGENLKIK Oenococcus Lys44 YTVRSGDTLGAIAAK SEQ IDNO:83  9.58 335–378  432 AF047001 Lysin oeni ØfOg44 YGTTYQKLASLNGIGSPYIIIPGEKLKVS YKVASGDTLSAIASK SEQ ID NO:84  9.95 389–432YGTSVSKLVSLNGLK NANYIYVGQTLRIK Thermotoga TM0409 YKVQKNDTLYSISLN SEQ IDNO:85  5.49  26–69  271 AE001720 maritime FGISPSLLLDWNPGL DPHSLRVGQEIVIPYTVKKGDTLDAIAKR SEQ ID NO:86  9.73  76–118 FFTTATFIKEANQLK SYTIYAGQKLFIPTM1686 HVVKRGETLWSIANQ SEQ ID NO:87  8.76 212–255  395 AE001809YGVRVGDIVLINRLE DPDRIVAGQVLKIG Treponema dniR HTIRSGDTLYALARR SEQ IDNO:88 10.58 607–650  779 AE001237 membrane- pallidum YGLGVDTLKAHNRAHbound lytic SATHLKIGQKLIIP murein trans- glycosylase D HVVQQGDTLWSLAKRSEQ ID NO:89  4.81 734–777 YGVSVENLAEENNLA VDATLSLGMILKTP TP0155YEVREGDVVGRIAQR SEQ ID NO:90  9.58  87–130  371 AE001200 YDISQDAIISLNKLRSTRALQVGQLLKIP TP0444 HVIAKGETLFSLSRR SEQ ID NO:91 10.98  67–110  342AE001221 YGVPLSALAQANNLA NVHQLVPGQRIVVP Borrelia BB0262 HKIKPGETLSHVAARSEQ ID NO:92  9.72 183–226(6)  417 AE001137 Hypothetical burgdorferiYQITSETLISFNEIK protein DVRNIKPNSVIKVP YIVKKNDSISSIASA SEQ ID NO:93 4.58 233–275 YNVPKVDILDSNNLD NEVLFLGQKLFIP *BB0625 YKVVKGDTLFSIAIK SEQID NO:94 10.02  44–86(28)  697 AE001164 N-acetylmura- YKVKVSDLKRINKLNmoyl-L- VDNIKAGQILIIP alanine amidase YTAKEGDTIESISKL SEQ ID NO:95  5.00115–157(7) VGLSQEEIIAWNDLR SKDLKVGMKLVLT YMVRKGDSLSKLSQD SEQ ID NO:96 9.20 165–207(8) FDISSKDILKFNFLN DDKLKIGQQLFLK HYVKRGETLGRIAYI SEQ IDNO:97 10.05 216–258(27) YGVTAKDLVALNGNR AINLKAGSLLNVL HSVAVGETLYSIARHSEQ ID NO:98  7.41 286–328 YGVLIEDLKNWNNLS SNNIMHDQKLKIF BB0761YKVKKGDTFFKIANK SEQ ID NO:99  9.34  59–102  295 AE001176 INGWQSGIATINLLDSPAVSVGQEILIP Lactobacillus *lys YTVVSGDSWNKIAQR SEQ ID NO:100  9.83399–442  442 X90511 lysin Øgle NGLSMYTLASQNGKS IYSTIYPGNKLIIK Bacillus*lytE IKVKKGDTLWDLSRK SEQ ID NO:101  9.55  28–70(17)  334 U38819.1D-Glutamate- subtilis YDTTISKIKSENHLR M-diaminopim- SDIIYVGQTLSIN lateendo- peptidase YKVKSGDSLWKISKK SEQ ID NO:102 10.16  88–130(20)YGMTINELKKLNGLK SDLLRVGQVLKLK YKVKSGDSLSKIASK SEQ ID NO:103 10.03151–193 YGTTVSKLKSLNGLK SDVIYVNQVLKVK spoVID CIVQQEDTIERLCER SEQ IDNO:104  4.20 525–568  575 P37963 stage VI YEITSQQLIRMNSLA sporulationLDDELKAGQILYIP protein D yaaH MVKQGDTLSAIASQY SEQ ID NO:105  3.89  1–43(5)  427 P37531 Hypothetical RTTTNDITETNEIPN protein PDSLVVGQTIVIPYDVKRGDTLTSIARQ SEQ ID NO:106 10.32  49–92 FNTTAAELARVNRIQLNTVLQIGFRLYIP yhdD IKVKSGDSLWKLAQT SEQ ID NO:107  9.56  29–71(22)  488Y14079 Hypothetical YNTSVAALTSANHLS protein TTVLSIGQTLTIPYTVKSGDSLWLIANE SEQ ID NO:108  9.62  94–136(39) FKMTVQELKKLNGLSSDLIRAGQKLKVS YKVQLGDSLWKIANK SEQ ID NO:109  9.72 176–218(23)VNMSIAELKVLNNLK SDTIYVNQVLKTK YTVKSGDSLWKIANN SEQ ID NO:110  9.65242–284(24) YNLTVQQIRNINNLK SDVLYVGQVLKLT YTVKSGDSLWVIAQK SEQ ID NO:111 9.72 309–351 FNVTAQQIREKNNLK TDVLGVGQKLVIS yojL IKVKSGDSLWKLSRQ SEQ IDNO:112  9.93  29–71(18)  414 Z99114 similar to YDTTISALKSENKLK cell wallSTVLYVGQSLKVP binding protein YTVAYGDSLWMIAKN SEQ ID NO:113  9.81 90–132(26) HKMSVSELKSLNSLS SDLIRPGQKLKIK YTVKLGDSLWKIANS SEQ ID NO:114 9.27 159–201(25) LNMTVAELKTLNGLT SDTLYPKQVLKIG YKVKAGDSLWKIANR SEQ IDNO:115  9.84 227–269 LGVTVQSIRDKNNLS SDVLQIGQVLTIS yocH ITVQKGDTLWGISQKSEQ ID NO:116  9.25  28–70(9)  287 AF027868 similar NGVNLKDLKEWNKLT topapQ SDKIIAGEKLTIS YTIKAGDTLSKIAQK SEQ ID NO:117  9.64  80–122FGTTVNNLKVWNNLS SDMIYAGSTLSVK ykvP HHVTPGETLSIIASK SEQ ID NO:118  8.65345–387  399 Z99111 Hypothetical YNVSLQQLMELNHFK protein SDQIYAGQIIKIR*xlyB YHVKKGDTLSGIAAS SEQ ID NO:119  9.72 179–222  317 Z99110N-acetylmura- HGASVKTLQSINHIT moyl-L- DPNHIKIGQVIKLP alanine amidaseyrbA HIVQKGDSLWKIAEK SEQ ID NO:120  8.51   4–48  387 Z99118 similar toYGVDVEEVKKLNTQL spore coat SNPDLIMPGMKIKVP protein ydhD HIVGPGDSLFSIGRRSEQ ID NO:121  5.49   4–46  439 Z99107 YGASVDQIRGVNGLD ETNIVPGQALLIPykuD YQVKQGDTLNSIAAD SEQ ID NO:122  6.10   4–46  164 Z99111FRISTAALLQANPSL QAGLTAGQSIVIP ØPBSX *xlyA YVVKQGDTLTSIARA SEQ ID NO:123 4.65 161–204  297 P39800 N-acetylmura- FGVTVAQLQEWNNIE moyl-L-DPNLIRVGQVLIVS alanine amidase ØPZA *orf15 YKVKSGDNLTKIAKK SEQ ID NO:12410.23 163–207(6)  258 P11187 HNTTVATLLKLNPSI KDPNMIRVGQTINVT (=Ø.29)HKVKSGDTLSKIAVD SEQ ID NO:125 10.17 214–258 P07540 NKTTVSRLMSLNPEITNPNHIKVGQTIRLS ØB103 *orfL5 HVVKKGDTLSEIAKK SEQ ID NO:126 10.11165–209(9)  263 X99260 lysozyme IKTSTKTLLELNPTI KNPNKIYVGQRINVGYKIKRGETLTGIAKK SEQ ID NO:127 10.61 219–263 NKTTVSQLMKLNPNIKNANNIYAGQTIRLK sphaericus *Pep1 ILIRPGDSLWYFSDL SEQ ID NO:128  8.86  3–46(6)  396 X69507 carboxypepti- FKIPLQLLLDSNRNI dase INPQLLQVGQRIQIP YTITQGDSLWQIAQN SEQ ID NO:129  7.15  53–96KNLPLNAILLVNPEI QPSRLHIGQTIQVP Salmonella nlpD YTVKKGDTLFYIAWI SEQ IDNO:130  8.64 121–164  377 AJ006131 dublin TGNDFRDLAQRNSIS APYSLNVGQTLQVGEscherichia *yebA YVVSTGDTLSSILNQ SEQ ID NO:131  4.13  77–121  419p24204 homologous to coli YGIDMGDISQLAAAD endopeptidase KELRNLKIGQQLSWTof Staphylo- coccus mltD YTVRSGDTLSSIASR SEQ ID NO:132 10.58 343–385(16) 452 P23931 MEMBRANE- LGVSTKDLQQWNKLR BOUND LYTIC GSKLKPGQSLTIG MUREINTRANS- GLYCOSYLASE D PRECURSOR YRVRKGDSLSSIAKR SEQ ID NO:133 10.18402–443 HGVNIKDVMRWNSDT ANLQPGDKLTLF UUG YTVKRGDTLYRISRT SEQ ID NO:13410.16  50–93  259 U28375 Hypothetical TGTSVKELARLNGIS proteinPPYTIEVGQKLKLG nlpD YTVKKGDTLFYIAWI SEQ ID NO:135  8.64 123–166  379P33648 Lipoprotein TGNDFRDLAQRNNIQ APYALNVGQTLQVG Drosophila Q9VNA1YTVGNRDTLTSVAAR SEQ ID NO:136  7.15 329–371 1325 AF125384 Lethal 82FDmelanogaster FDTTPSELTHLNRLN protein SSFIYPGQQLLVP Drosophila Q961P8YTVGNRDTLTSVAAR SEQ ID NO:136  7.15 104–146  678 AAK92873 melanogasterFDTTPSELTHLNRLN SSFIYPGQQLLVP Caenorhabditis F43G9.2 RKVKNGDTLNKLAIK SEQID NO:137 10.01  12–55  179 Z79755 elegans YQVNVAEIKRVNNMVSEQDFMALSKVKIP Caenorhabditis F52E1.13 YTITETDTLERVAAS SEQ ID NO:138 7.08  24–66  819 U41109 elegans HDCTVGELMKLNKMA SRMVFPGQKILVPCaenorhabditis F07G11.9 TEIKSGDSCWNIASN SEQ ID NO:139  8.32  23–66(11)1614 U64836/ Putative elegans AKISVERLQQLNKGM AF016419 EndochitinaseKCDKLPLGDKLCLA LKLKAEDTCFKIWSS SEQ ID NO:140  7.84  78–121(21)QKLSERQFLGMNEGM DCDKLKVGKEVCVA HKIQKGDTCFKIWTT SEQ ID NO:141  8.65143–186(21) NKISEKQFRNLNKGL DCDKLEIGKEVCIS LKIKEGDTCYNIWTS SEQ ID NO:142 4.54 208–251(19) QKISEQEFMELNKGL DCDKLEIGKEVCVT YRFKKGDTCYKIWTS SEQ IDNO:143  9.35 271–314(20) HKMSEKQFRALNRGI DCDRLVPGKELCVG ITVKPGDTCFSIWTSSEQ ID NO:144  4.21 335–378(23) QKMTQQQFMDINPEL DCDKLEIGKEVCVTVKINPGDTCFNIWTS SEQ ID NO:145  6.30 402–445(21) QRMTQQQFMDLNKRLDCDKLEVGKEVCVA VQINPGDTCFKIWSA SEQ ID NO:146  4.60 467–510(37)QKLTEQQFMELNKGL DCDRLEVGKEVCIA TEVKEGDTCFKIWSA SEQ ID NO:147  5.12548–591(44) HKITEQQFMEMNRGL DCNRLEVGKEVCIV IKVKEGDTCFKIWSA SEQ ID NO:148 7.85 636–679(66) QKMTEQQFMEMNRGL DCNKLMVGKEVCVS ATITPGNTCFNISVA SEQ IDNO:149  3.99 746–786(8) YGINLTDLQKTYDCK ALEVGDTICVS IEVIKGDTCWFLENA SEQID NO:150  4.67 795–838 FKTNQTEMERANEGV KCDNLPIGRMMCVW CaenorhabditisT01C4.1 HTIKSGDTCWKIASE SEQ ID NO:151  5.01  23–66(51) 1484 U70858Putative elegans ASISVQELEGLNSKK Endochitinase SCANLAVGLSEQEFIHVKEGDTCYTIWTS SEQ ID NO:152  4.12 118–161(25) QHLTEKQFMDMNEELNCGMLEIGNEVCVD ATVTPGSSCYTISAS SEQ ID NO:153  3.07 187–226(9)YGLNLAELQTTYNCD ALQVDDTICVS IEILNGDTCGFLENA SEQ ID NO:154  3.85 236–279FQTNNTEMEIANEGV KCDNLPIGRMMCVW Bacillus #ypbE HTVQKKETLYRISMK SEQ IDNO:155  9.45 191–136  240 L47648 subtilis YYKSRTGEEKIRAYNHLNGNDVYTGQVLDI P Citrobacter #eae YTLKTGESVAQLSKS SEQ ID NO:156  8.59 65–113  936 Q07591 fruendii QGISVPVIWSLNKHL YSSESEMMKASPGQQ IILPEscherichia #eae YTLKTGETVADLSKS SEQ ID NO:157  5.65  65–113  934 P43261Necessary coli QDINLSTIWSLNKHL for close YSSESEMMKAAPGQQ (intimate) IILPattachment of bacteria Micrococcus #rpf IVVKSGDSLWTLANE SEQ ID NO:158 3.85 171–218  220 Z96935 Bacterial luteus YEVEGGWTALYEANK CytokineGAVSDAAVIYVGQEL VL Bacillus #yneA IEVQQGDTLWSIADQ SEQ ID NO:159  3.81 40–90  105 Z73234 subtilis VADTKKINKNDFIEW VADKNQLQTSDIQPG DELVIPStreptococcus # YTVKYGDTLSTIAEA SEQ ID NO:160  4.23  47–103  393 U09352pyogenes MGIDVHVLGDINHIA NIDLIFPDTILTANY NQHGQATTLT Bacillus #xkdPYTVKKGDTLWDIAGR SEQ ID NO:161 11.23 176–234 235 p54335 subtilisFYGNSTQWRKIWNAN KTAMIKRSKRNIRQP GHWIFPGQKLKIP Bacillus #yqbPYTVKKGDTLWDIAGR SEQ ID NO:161 11.23 177–234 235 G1225954 subtilisFYGNSTQWRKIWNAN KTAMIKRSKRNIRQP GHWIFPGQKLKIP Bacillus # YTVKKGDTLWDLAGKSEQ ID NO:162 10.75 161–218 219 P45932 subtilis FYGDSTKWRKIWKVNKKAMIKRSKRNIRQP GHWIFPGQKLKIP a) Proteins listed were obtained by ahomology search in the SWISSPROT, PIR, and Genbank databases with therepeats of AcmA using the BLAST program. b) *; genes encoding cell wallhydrolases. #; proteins containing repeats that are longer than theconsensus sequence. c) The number of aa residues between the repeats aregiven between brackets. d) Number of aa of the primary translationproduct. e) Genbank accession number. Consensus repeatYxVKxGDTLxxIAxxxxxxxxxLxxxNxxLxxxxxIxxGQxIxVx (SEQ ID NO:163)                 H IR  ESV  LS         I      I     L     L I                   L     I  V                       V     V L

TABLE 4 Calculated pI's of individual repeat sequences of the AcmA andAcmD protein anchors. AcmA anchor domain AcmD anchor domain CalculatedCalculated Repeat pI Repeat pI A1 9.75 D1 4.15 A2 9.81 D2 3.78 A3 10.02D3 4.15 A1A2A3 10.03 D1D2D3 3.85

TABLE 5 Hybrid protein anchors composed of different AcmA and AcmDrepeat sequences and their calculated pI's. Composition of hybridsCalculated AcmA-repeat sequence AcmD-repeat sequence pI A1A2A3 — 10.03A1A2 D1 9.53 A1A2A3 D1D2D3 8.66 A1 D2 8.45 A3 D1D2 7.39 A1A2 D1D2D3 6.08A3 D1D2D3 5.07 A1 D1D2D3 4.37 — D1D2D3 3.85

1. A method for binding a proteinaceous substance to cell-wall materialof a Gram-positive bacterium, said method comprising: treating saidcell-wall material with an acid solution, thus removing a cell-wallcomponent selected from the group consisting of a protein, alipoteichoic acid, a carbohydrate, and combinations thereof from saidcell-wall material, thus producing spherical peptidoglycanmicroparticles; and contacting said spherical peptidoglycanmicroparticles with a proteinaceous substance, thus binding saidproteinaceous substance to said spherical peptidoglycan microparticles;wherein said proteinaceous substance comprises an immunogenicdeterminant and an AcmA cell-wall binding domain, homolog, or functionalderivative thereof that binds to the treated cell-wall material.
 2. Themethod according to claim 1, wherein said acid solution comprises anacid selected from the group consisting of acetic acid (HAc),hydrochloric acid (HCI), sulphuric acid (H₂SO₄), trichloro acetic acid(TeA), trifluoro acetic acid (TFA), monochloro acetic acid (MCA), andmixtures thereof.
 3. The method according to claim 1, wherein saidGram-positive bacterium is selected from the group consisting of aLactobacillus spp., a Lactococcus spp., a Bacillus spp., and aMycobacterium spp.
 4. The method according to claim 1, wherein saidimmunogenic determinant is of a pathogen origin.
 5. The method accordingto claim 4, wherein said pathogen is Plasmodium falciparum.
 6. Themethod according to claim 5, wherein said proteinaceous substance is amerozoite surface antigen 2 fused to a lactococcal AcmA protein anchor.7. The method according to claim 4, wherein said pathogen isStreptococcus pneumornae.
 8. The method according to claim 7, whereinsaid proteinaceous substance is a Streptococcal proteinase maturationprotein A antigen fused to a lactococcal AcmA protein anchor.
 9. Amethod for binding a proteinaceous substance to cell-wall material of aGram-positive bacterium, said proteinaceous substance comprising an AcmAcell wall binding domain, homolog, or functional derivative thereofcapable of binding to said cell-wall material of the Gram-positivebacterium, said method comprising: treating said cell-wall material withan acid solution, thus removing a cell-wall component selected from thegroup consisting of a protein, a lipoteichoic acid, a carbohydrate, andcombinations thereof from said cell-wall material, thus producingspherical peptidoglycan microparticles; and contacting said sphericalpeptidoglycan microparticles with said proteinaceous substance, so as tobind said proteinaceous substance to said spherical peptidoglycanmicroparticles.
 10. The method according to claim 9, wherein saidproteinaceous substance is contacted with said spherical peptidoglycanmicroparticles at a pH that is lower than the calculated pI value ofsaid AcmA cell wall binding domain, homolog, or functional derivativethereof.
 11. The method according to claim 1, wherein said Gram-positivebacterium is a Lactococcus species.
 12. A method for binding aproteinaceous substance to cell-wall material of a Gram-positivebacterium, said method comprising: treating the Gram-positive bacteriumwith an acid solution; and contacting the acid treated Gram-positivebacterium with a heterologous protein and means for attaching theheterologous polypeptide to the acid treated Gram-positive bacterium.13. The method according to claim 12, wherein the means for attachingthe heterologous polypeptide comprises an AcmA cell-wall binding domain.14. The method according to claim 12, wherein the heterologous proteincomprises a Streptococcal proteinase maturation protein A antigen andthe means for attaching the heterologous polypeptide comprises alactococcal AcmA protein anchor.
 15. The method according to claim 9,further comprising isolating the spherical peptidoglycan microparticles.16. The method according to claim 10, further comprising isolating thespherical peptidoglycan microparticles.
 17. A composition produced bythe method according to claim
 1. 18. The method according to claim 12,wherein the heterologous protein comprises a merozoite surface antigen 2and the means for attaching the heterologous polypeptide comprises alactococcal AcmA protein anchor.
 19. The method according to claim 1,further comprising isolating said spherical peptidoglycanmicroparticles.